Egfr-directed car therapy for glioblastoma

ABSTRACT

Glioblastoma (GB) remains the most aggressive primary brain malignancy; brain metastasis, such as breast cancer brain metastases (BCBMs), are also aggressive and are associated with poor prognosis. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered cells to treat brain cancers has not been explored. The present disclosure presents compostions and methods for using CAR expressing cells in the treatment of various cancers, including brain cancers such as GB and BCBMs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/143,744, filed Apr. 6, 2015, the contentof which is hereby incorporated by reference in its entirety.

BACKGROUND

Brain cancers are dangerous and difficult to treat. Glioblastoma (GB) isthe most common and the most aggressive primary brain tumor. Cancer ofthe brain also poses a risk where it is the result of metastasis fromanother source; for example, breast cancer brain metastases (BCBMs) arecommon in patients with metastatic breast cancer. Even withchemotherapy, radiation, and surgical resection, the median overallsurvival of GB patients is only 14.6 months (Morgan, R. A. et al. (2012)Hum Gene Ther. 23:1043-1053). BCBMs are likewise highly resistant toavailable treatment and indicate poor prognosis for the patient.Conventional therapies generally lack specificity and can cause damageto the surrounding brain parenchyma and systemic tissues, a factor thatlimits their use (Imperato, J. P. et al. (1990) Ann Neurol. 28:818-822).Immune-based therapies for GB are a promising alternative toconventional treatments with a potential long-term benefit of generatinga sustainable anti-tumor response with potential to target bothlocalized and infiltrating tumor cells (Mellman, I. et al. (2011) Nature480:480-489). The epidermal growth factor receptor (EGFR) plays animportant role in various tumors including GB. EGFR is the mostfrequently amplified gene in GB, while its expression in normal braintissue is either undetectable or extremely low (Parsons, D. W. et al.(2008) Science 321:1807-1812; Salomon, D. S. et al. (1995) Crit. RevOncol Hematol. 19:183-232). Binding of ligand to EGFR leads to receptorhomo- and heterodimers formation, autophosphorylation of several keytyrosine residues leading to activation of several intracellulardownstream signaling pathways including the Ras/Raf/MEK/ERK pathway, thePLCγ-PKC pathway and the PI3K/AKT pathway, resulting in cellproliferation, motility and survival (Zandi, R. et al. (2007) CellSignal 19:2013-2023). Approximately 20-40% of EGFR-amplified tumorsharbor the EGFR variant III mutant (EGFRvIII), which contains a deletionof exons 2-7 in the extracellular ligand-binding domain (Aldape, K. D.et al. (2004) Journal of Neuropathology and Experimental Neurology63:700-707; Biernat, W. et al. (2004) Brain Pathol. 14:131-136; Fan, Q.W. et al. (2013) Cancer Cell 24:438-449; Sugawa, N. et al. (1990) ProcNatl Acad Sci. USA 87:8602-8606). This mutant form shows constitutiveactivation in the absence of ligand to activate the tumor-promotingsignaling pathways (Ohno, M. et al. (2010) Cancer Sci. 101:2518-2524).

SUMMARY OF THE DISCLOSURE

Glioblastoma (GB) remains the most aggressive primary brain malignancy.Adoptive transfer of chimeric antigen receptor (CAR)-modified immunecells has emerged as a promising anti-cancer approach, yet the potentialutility of CAR-engineered natural killer (NK) cells to treat GB has notbeen explored. Tumors from approximately 50% of GB patients expresswild-type EGFR (wtEGFR) and in fewer cases express wtEGFR and the mutantform EGFRvIII; however, previously reported CAR T cell only focuses ontargeting EGFRvIII.

Aspects of the disclosure relate to a chimeric antigen receptor (CAR)comprising: (a) an antigen binding domain; (b) a hinge domain; (c) atransmembrane domain; and (d) an intracellular domain. This disclosureprovides a novel CAR—an Epidermal Growth Factor Receptor (“EGFR”)chimeric antigen receptor (CAR) comprising, or alternatively consistingessentially of, or yet further consisting of: (a) an antigen bindingdomain of an anti-EGFR antibody that recognized both wild type and/ormutant Epidermal Growth Factor Receptor (EGFR) (“wt EGFR and mutantEGFR”); (b) a hinge domain polypeptide; (c) a costimulatory polypeptide;and (d) a CD3 zeta signaling domain. In one aspect, the costimulatorymolecule comprises an intracellular domain and a transmembrane domain.Non-limiting examples include CD8, a 4-1BB costimulatory signalingregion, a CD28 costimulatory molecule, OX40, ICOS and CD27. In oneaspect, the antigen binding domain of the anti-EGFR antibody comprisesan anti-EGFR heavy chain (HC) variable region and an anti-EGFR lightchain (LC) variable region. In a yet further aspect, the EGFR CARfurther comprises, or alternatively consists essentially of, or yetfurther consists of a linker polypeptide located between the anti-EGFRHC variable region and the anti-EGFR LC variable region. The CAR canfurther comprise a detectable label or a purification marker.

Polynucleotides encoding the EGFR CARs and their complements andequivalents of each are further disclosed herein. Vectors and host cellscontaining the polynucleotides and/or EGFR CARs are provided herein. Thevectors are plasmids or vectors. The cells can be prokaryotic oreukaryotic cells. In one aspect, the cells are T cells or NK cells. Thecells can be expanded and therefore an expanded population of the cellsis further provided herein. The cells are, in one aspect, an activatedpopulation of cells such as T cells.

Diagnostic and therapeutic use of these compositions are furtherprovided herein. For example, provided is method of inhibiting thegrowth of one or more of a cell and/or tumor expressing wt EGFR ormutant EGFR, or wt EGFR and mutant EGFR on the surface of the cell or astem cell expressing EGFR, by contacting these cells with an effectiveamount of an isolated cell comprising an EGFR CAR as described herein.The cell can be a tumor cell, e.g., a glioblastoma or a cancer stemcell, e.g., a glioblastoma stem cell. The contacting can be in vitro orin vivo. When practiced in vitro, the method is useful to screen for newtherapies or combination therapies. In vivo, the method is useful totreat a patient suffering from a cancer that expresses EGFR, e.g., abrain cancer such as glioblastoma or metastasis of another cancer (e.g.breast cancer) in the brain.

In another aspect, this disclosure provides a method of inhibiting thegrowth of one or more of: a cell expressing wt EGFR or mutant EGFR, orwt EGFR and mutant EGFR, on the surface of the cell in a subject in needthereof. This method comprises, or alternatively consisting essentiallyof, or yet further consisting of, administering to the subject aneffective amount of the isolated cell comprising the EGFR CAR asdescribed herein. The cell expressing EGFR can be a tumor cell or acancer stem cell, e.g., a glioblastoma or a glioblastoma stem cell. Theisolated cells expressing the EGFR CAR can be autologous to the subjectreceiving the cells. Yet further provided are methods to treat a cancerin a subject in need thereof, wherein the cancer cell expresses wt EGFRor mutant EGFR, or wt EGFR and mutant EGFR, by administering to thesubject an effective amount the isolated cell comprising the EGFR CAR asdescribed herein. The isolated cells of these methods can be NK cellsand/or T-cells and can be autologous to the subject being treated. In afurther aspect, the cells are administered by any appropriate route asdetermined by the treating physician and include without limitation,intracranial injection, intravenous administration

Yet further provided is an isolated complex comprising the EGFR CAR asdescribed herein and an EGFR protein or a fragment thereof and/or mutantEGFR protein or a fragment thereof. In another aspect, provided hereinis an isolated complex comprising the EGFR CAR as described herein and acell expressing a wt EGFR or mutant EGFR, or wt EGFR and mutant EGFR.

Further aspects of the disclosure relate to compositions and combinationcancer therapy with the EGFR CAR expressing cells, such as T cells or NKcells, described herein and oncolytic herpes simplex viruses. Someembodiments relate to a method of treating a tumor or cancer where botha EGFR CAR expressing cell and an oncolytic herpes simplex areadministered to a patient in need thereof. In some embodiments, the EGFRCAR expressing cells are administered simultaneously with the oncolyticherpes simplex virus; in other embodiments, the EGFR CAR expressingcells are administered before or after administration of the oncolyticherpes simplex virus.

Kits comprising one or more of the above compositions are furtherprovided. Instructions to use them diagnostically or therapeutically arefurther provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show expression of EGFR on GB and GB stem cells. (FIG. 1A)Surface EGFR expression on glioma cell lines (U251, LN229, andGli36dEGFR) and glioma stem cells (GB30, GB83, GB326, GB1123, GB84V3SL,GB157V3SL, and GB19) was monitored by flow cytometry. NK cell lines NKLand NK-92 serve as negative controls. (FIG. 1B). Determination of EGFRmRNA expression by RT-PCR in glioma cell lines and glioma stem cellsspecified in FIG. 1A. Flow plots and data are representative of threeindependent experiments.

FIGS. 2A-2B show generation of an EGFR-specific CAR and detection of itsexpression on CAR-transduced NK cells. (FIG. 2A) Schematicrepresentation of the EGFR-CAR lentiviral construct. (FIG. 2B)Expression of chimeric EGFR scFv on the surface of NK-92 and NKL cellstransduced either with the EGFR-CAR construct (NK-92-EGFR-CAR andNKL-EGFR-CAR, respectively) or the empty vector construct (EV) Cellswere FACS-sorted by GFP expression, then analyzed by flow cytometryafter the cells were stained with an anti-mouse Fab antibody (solidlines) or IgG isotype control (dashed lines). SP, signal peptide; VH,heavy chain; VL, light chains; ScFv, single chain variable fragment.Flow plots and data are representative of three independent experiments.

FIGS. 3A-3B show EGFR-CAR-modified NK-92 and NKL cells recognize andkill EGFR⁺ GB cell line cells. (FIG. 3A) Cytotoxic activity of emptyvector (EV)-transduced or EGFR-CAR-transduced NK-92 and NKL cellsagainst Gli36dEGFR, LN229 or U251 cells using a standard chromium-51release assay. (FIG. 3B) IFN-γ release of empty vector EV-transduced orEGFR-CAR-transduced NK-92 and NKL cells in the absence or presence ofGli36dEGFR, LN229 or U251 cells using ELISA assay. Representative dataof three independent experiments are shown. *p<0.05; **p<0.01.

FIGS. 4A-4B show EGFR-CAR-modified NK-92 and NKL cells display enhancedlysis of EGFR⁺ GB stem cells. (FIG. 4A) Cytotoxic activity of NK-92-EVor NK-92-EGFR-CAR cells (upper panel) and NKL-EV or NKL-EGFR-CAR cells(lower panel) against GB1123, GB30, GB157V3SL, and GB84V3SL GB stemcells using chromium-51 release assay. (FIG. 4B) ELISA analysis of IFN-γsecretion by NK-92-EGFR-CAR or NK-92-EV cells (upper panel) and NKL-EVor NKL-EGFR-CAR cells (lower panel) when co-cultured with GB stem cells.Representative data of three independent experiments are shown. *p<0.05;**p<0.01.

FIGS. 5A-5C show enhanced target recognition of NK-92-EGFR-CAR cellsdepends on expression of EGFR on cell surface. (FIG. 5A) Flow cytometryusing an anti-EGFR antibody (solid line) or IgG isotype control (dottedline) to detect EGFR expression on the surface of GB19 cells transfectedwith an empty vector or vectors containing wtEGFR or EGFRvIII. (FIG. 5B)Cytotoxicity of NK-92-EV or NK-92-EGFR-CAR (upper panel) and NKL-EV orNKL-EGFR-CAR (lower panel) against the GB19, GB19-wtEGFR, andGB19-EGFRvIII cells shown in FIG. 5A. The GB19 cells were incubated withNK-92 or NKL cells at various Effector/Target (E/T) ratios for 4 h.Tumor lysis was determined using chromium-51 release assay. (FIG. 5C)NKL-EV or NKL-EGFR-CAR (left) and NK-92-EGFR-CAR or NK-92-EV cells(right) were co-cultured with equal numbers of GB19-Vector or GB19-EGFRcells for 24 h. Supernatants were then harvested for measurement ofIFN-γ secretion using ELISA. Flow plots and data are representative ofthree independent experiments. *p<0.05, **p<0.01.

FIGS. 6A-6E show that NK-92-EGFR-CAR cells suppress in vivo growth oforthotopic human glioma stem cells, prolong the survival ofglioma-bearing mice, and localize in the brain without migrating toother organ and tissues. (FIG. 6A) Brain bioluminescence imaging of micebearing GB30 tumors. NSG mice were inoculated with luciferase-expressingGB30 cells via stereotaxic injection (day 0). Seven days afterinoculation, mice were intracranially infused once with emptyvector-transduced NK-92 cells (NK-92-EV), EGFR-CAR-transduced NK-92cells (NK-92-EGFR-CAR) or Hank's buffered salt solution (HBSS; negativecontrol). (FIG. 6B) Quantification summary of units of photons persecond per mouse from FIG. 6A. * indicates p<0.05. (FIG. 6C)GB30-bearing mice treated with NK-92-EGFR-CAR cells showed significantlyincreased overall survival compared to the mice treated with NK-92-EVcells or HBSS (** p<0.01), as determined by Kaplan-Meier survival curves(n=5 for each group). (FIG. 6D) Determination of presence ofCD56⁺CD3^(˜) human EGFR-CAR NK-92 cells by flow cytometry in liver,lung, blood, spleen, bone marrow (BM), and brain 3 days afterintracranial injection of the CAR NK cells into brain of GB30-bearingmice (FIG. 6E) Determination of EGFR-CAR expression by RT-PCR in liver,lung, blood, spleen, bone marrow (BM), and brain 3 days afterintracranial injection of the CAR NK cells into brain of GB30-bearingmice. NC=negative control (no DNA template was added), PC=positivecontrol, EGFR-CAR NK-92 cells. *p<0.05, **p<0.01.

FIGS. 7A-7C show that EGFR-CAR-transduced NK-92 cells inhibitwtEGFR-expressing GB tumor growth and prolong survival of tumor-bearingmice in an orthotopic xenograft GB model. (FIG. 7A) Brainbioluminescence imaging of mice bearing U251 tumors. NSG mice wereintracranially implanted with 10⁵ luciferase-expressing U251 cells viastereotaxic injection (day 0). Day 10, 40, 70 after inoculation, micewere intracranially infused with NK-92-EV cells, NK-92-EGFR-CAR cells orHBSS as negative control. Brain bioluminescence imaging of mice wastaken on Day 100. (FIG. 7B) Quantification summary of units of photonsper second per mouse from FIG. 7A. ** indicates p<0.01. (FIG. 7C)U251-bearing mice treated with NK-92-EGFR-CAR cells showed significantlyincreased overall survival compared to the mice treated with NK-92-EVcells (* p<0.05), or HBSS ** p<0.01), as determined by Kaplan-Meiersurvival curves (n=5 for each group).

FIGS. 8A-8B show that EGFR-CAR primary NK cells display enhancederadication of EGFR⁺ GB cells and patient-derived GB stem cells. (FIG.8A) EGFR-CAR-modified primary NK cells (primary NK-CAR) displayedaugmented cytolytic activity towards EGFR⁺ Gli36dEGFR and U251 GB celllines in comparison with mock-transduced primary NK cells (primaryNK-EV). (FIG. 8B) EGFR-CAR-modified NK cell (primary NK-CAR) showedenhanced cytotoxicity towards EGFR⁺-patient-derived GB30 and GB157V3SLstem cells in comparison with mock-transduced primary NK cells (primaryNK-EV). Data presented are representative of three experiments withsimilar results, examining NK cells isolated from different healthydonors. *p<0.05; **p<0.01.

FIGS. 9A-9C show that enhanced target recognition of NK-92-EGFR-CARcells depends on expression of EGFR on the cell surface. (FIG. 9A) Flowcytometric analysis using anti-EGFR antibody (solid line) or IgG isotypecontrol (dotted line) of 293T cells transduced with empty vector (EV;left), wtEGFR (center) or EGFRvIII (right). (FIG. 9B) Cytotoxicity ofNK-92-EV or NK-92-EGFR-CAR (top panel) and NKL-EV or NKL-EGFR-CAR (lowerpanel) against 293T-EV (left), 293T-EGFR (center), and 293T-EGFRvIII(right) cells. 293T cells were incubated with NK cells at variousEffector/Target (E/T) ratios for 4 h. Tumor lysis was determined usingchromium-51 release assay (FIG. 9C) After coincubation of target cellsand effector cells for 24 h, supernatants from the co-cultures weremeasured for IFN-γ secretion using ELISA. Data presented arerepresentative of three experiments with similar results. *p<0.05;**p<0.01.

FIGS. 10A-10B show that the effects of NK-92-EGFR-CAR cells are bluntedby an EGFR blocking antibody (Ab). (FIG. 10A) Cytotoxicity of NK-92-EVor NK-92-EGFR-CAR against GB30 cells (Left) or U251 cells (Right)pretreated with an EGFR-specific monoclonal antibody 528 or anIgG-matched isotype control antibody. Target cells were incubated withpre-treated NK-92-EV or NK-92-EGFR-CAR cells at various Effector/Target(E/T) ratios for 4 h. Tumor lysis was determined using chromium-51release assay. (FIG. 10B) After co-incubation of target cells andeffector cells for 24 h, supernatants from the co-cultures were measuredfor IFN-γ secretion using ELISA. Data presented are representative ofthree experiments with similar results. *p<0.05; **p<0.01.Ab=EGFR-specific monoclonal antibody 528; iso=IgG-matched isotypecontrol antibody.

FIG. 11 shows that NK-92-EGFR-CAR cells are located in tumor area afterintratumoral injection. NK-92-EGFR-CAR cells were intratumorallyinjected into mouse brains seven days after GB30 implantation. The mousebrains were harvested 3 days later, embedded by paraffin, and processedfor Hematoxylin and Eosin (H&E) staining. H&E staining showed thatNK-92-EGFR-CAR cells only existed inside tumor area. Magnifications of20×, 40× and 100× were shown (Objective. 2×, 4× or 10×; Eyepiece: 10×).

FIGS. 12A-12B depict expression of EGFR in breast cancer cell lines andtissues. Expression of EGFR on the cell surface of breast cancer celllines (MDA-MB-231, MDA-MB-468, and MCF-7) was detected by flow cytometry(FIG. 12A). Hematoxylin and eosin (HE) staining and immunohistochemistry(IHC) of EGFR expression was performed for tumor tissues from patientswith primary breast cancer and brain metastases (FIG. 12B).

FIGS. 13A-13E demonstrate that EGFR-CAR NK-92 cells recognize and lyseEGFR positive cells of breast cancer cell lines. Expression of EGFR scFvon EGFR-CAR-transduced NK-92 cells, was determined by flow cytometryusing a goat anti-mouse F(ab′)₂ polyclonal antibody (FIG. 13A) IFN-γrelease by empty vector (EV)-transduced or EGFR-CAR-transduced NK-92cells in the absence or presence of MDA-MB-231, MDA-MB-468 or MCF-7cells was determined using a standard ELISA assay. **P<0.01 (FIG. 13B).Cytotoxic activity of empty vector (EV)-transduced orEGFR-CAR-transduced NK-92 cells against MDA-MB-231 (FIG. 13C),MDA-MB-468 (FIG. 13D), or MCF-7 (FIG. 13E) cells was performed using astandard chromium-51 release assay. (E, effect cell; T, target cell)

FIGS. 14A-14D depict enhanced cytotoxicity and IFN-γ production ofEGFR-CAR primary NK cells when stimulated with EGFR⁺ breast cancercells. IFN-γ release by empty vector (EV)-transduced orEGFR-CAR-transduced primary NK cells in the absence or presence ofMDA-MB-231, MDA-MB-468 or MCF-7 cells was determined using a standardELISA assay (FIG. 14A). Cytotoxic activity of empty vector(EV)-transduced or EGFR-CAR-transduced primary NK cells againstMDA-MB-231 (FIG. 14B), MDA-MB-468 (FIG. 14C), or MCF-7 (FIG. 14D) cellswas performed using a standard chromium-51 release assay. (E, effectcell; T, target cell)

FIGS. 15A-15C show that oHSV-1 alone can lyse and eradicate breastcancer cell line tumor cells. Dose-dependent cytotoxicity of oHSV-1 tobreast cancer cell lines (MDA-MB-231, MDA-MB-468 or MCF-7) afterco-culture for 48 h was detected by MTS *P<0.05; **P<0.01 (FIG. 15A).FIG. 15B depicts MTS assays of oHSV-1 cytotoxicity against breast cancercell lines, MDA-MB-231, MDA-MB-468 or MCF-7, after co-cultured of themfor different time periods. FIG. 15C shows measurement of luciferaselevels in the media of the co-culture of MDA-MB-231-CBRluc-EGFP cellsand oHSV-1.

FIGS. 16A-16B reveal that the combinational treatment of EGFR-CAR NK-92cells and oHSV-1 results in more efficient eradication of breast cancertumor cells in vitro. Tumor cells were treated with CAR cells alone,oHSV-1 alone, EGFR-CAR NK-92 cells for 4 h followed by oHSV-1(CAR+oHSV), or oHSV for 4 h followed by EGFR-CAR NK-92 cells (oHSV+CAR).Eradication of MDA-MB-231 tumor cells expressing CBRluc-EGFP wasmeasured by luciferase release to supernatants at different time points(FIG. 16A). Regardless of the order, the EGFR-CAR NK-92 cells incombination with oHSV-1 (CAR+oHSV or oHSV+CAR) eradicated moreMDA-MB-231 tumor cells than EGFR-CAR NK-92 cells alone (CAR) or oHSV-1alone (oHSV), determined by the relative light units of luciferaseremained in the MDA-MB-231-CBRluc-EGFP cells on day 4 after co-cultured.**P<0.01 (FIG. 16B). Data are representative of three independentexperiments

FIGS. 17A-17B demonstrate that EGFR-CAR transduced NK-92 cells inhibitMDA-MB-231 tumor growth with prolonged survival of the tumor-bearingmice. Brain bioluminescence imaging of mice bearing BCBM tumors. NSGmice were inoculated with MDA-MB-231-CBRluc-EGFP cells via stereotaxicinjection (day 0). 10 days after inoculation, mice were intracraniallyinfused once with EGFR-CAR NK-92, oHSV-1, NK-92-EV, or HBSS. The mice ofcombined treatment group were injected with oHSV-1 on day 15. Four weeksafter inoculation with MDA-MB-231-CBRluc-EGFP cells, the mice wereintraperitoneally infused with D-luciferin and imaged using the In VivoImaging System (FIG. 17A). MDA-MB-231-CBRluc-EGFP tumor-bearing micewere intratumorally treated with EGFR-CAR NK-92 cells followed by oHSV-1injection (CAR+oHSV), EGFR-CAR NK-92 cells alone (CAR), oHSV-1 alone, orHBSS control. As a result, EGFR-CAR NK-92 cells followed by oHSV-1injection showed significantly increased overall survival than the restof treatments as determined by Kaplan-Meier survival curves (n=5 foreach group) (FIG. 17B).

FIG. 18 shows the transduction efficiency of lentiviruses in humanprimary NK cells. The percentage of GFP (+) cells was determined by flowcytometry after human primary NK cells were infected with EGFR-CARlentiviruses.

FIG. 19 shows the change in expression of activation markers on thesurface of EGFR-CAR NK-92 cells in response to breast cancer cells.Surface expression of CD27 and CD69 in EGFR-CAR NK-92 cells wasdetermined after co-culture with breast cancer cells (MDA-MB-231,MDA-MB-468 and MCF-7) overnight by flow cytometry.

FIGS. 20A-20B demonstrate lysis of breast cancer cells by oHSV alone.Lysis of breast cancer cell line (MDA-MB-231) by oHSV after co-culturefor 4 days, demonstrated by the bright images under microscope (FIG.20A). EGFR-CAR NK92 cells were treated with oHSV-1 for 4 days, andmicroscopic examination showed that oHSV-1 had no obvious effect on theproliferation and viability of EGFR-CAR NK-92 cells (FIG. 20B).

FIG. 21 depicts lysis of breast cancer cell line (MDA-MB-231) by oHSV,EGFR-CAR NK-92 cells, and their combination. “MDA-MB-231+CAR+oHSV”denotes treatment of EGFR-CAR NK-92 cells for 4 h, followed by oHSV-1treatment. “MDA-MB-231+oHSV+CAR” denotes treatment of oHSV-1 for 4 h,followed by treatment of EGFR-CAR NK-92 cells.

FIG. 22 shows quantification of emitted photons from intracranialxenograft GB tumors treated with vehicle, NK-92 cells, oHSV, EGFR-CARNK-92 cells, and the combination of oHSV and EGFR-CAR NK-92 cells.

DETAILED DESCRIPTION

It is to be understood that the present disclosure is not limited toparticular aspects described, as such may, of course, vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular aspects only, and is not intended to be limiting,since the scope of the present disclosure will be limited only by theappended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this technology belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present technology, the preferredmethods, devices and materials are now described. All technical andpatent publications cited herein are incorporated herein by reference intheir entirety. Nothing herein is to be construed as an admission thatthe present technology is not entitled to antedate such disclosure byvirtue of prior invention.

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology, and recombinant DNA,which are within the skill of the art. See, e.g., Sambrook and Russelleds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson el al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); and Herzenberg et al. eds (1996) Weirs Handbook of ExperimentalImmunology.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate, oralternatively by a variation of +/−15%, or alternatively 10%, oralternatively 5%, or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

It is to be inferred without explicit recitation and unless otherwiseintended, that when the present technology relates to a polypeptide,protein, polynucleotide or antibody, an equivalent or a biologicallyequivalent of such is intended within the scope of the presenttechnology.

Definitions

As used in the specification and claims, the singular form “a”, “an”,and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “animal” refers to living multi-cellularvertebrate organisms, a category that includes, for example, mammals andbirds. The term “mammal” includes both human and non-human mammals.

The terms “subject,” “host,” “individual,” and “patient” are as usedinterchangeably herein to refer to human and veterinary subjects, forexample, humans, animals, non-human primates, dogs, cats, sheep, mice,horses, and cows. In some embodiments, the subject is a human.

As used herein, the term “antibody” collectively refers toimmunoglobulins or immunoglobulin-like molecules including by way ofexample and without limitation, IgA, IgD, IgE, IgG and IgM, combinationsthereof, and similar molecules produced during an immune response in anyvertebrate, for example, in mammals such as humans, goats, rabbits andmice, as well as non-mammalian species, such as shark immunoglobulins.Unless specifically noted otherwise, the term “antibody” includes intactimmunoglobulins and “antibody fragments” or “antigen binding fragments”that specifically bind to a molecule of interest (or a group of highlysimilar molecules of interest) to the substantial exclusion of bindingto other molecules (for example, antibodies and antibody fragments thathave a binding constant for the molecule of interest that is at least10³ M⁻¹ greater, at least 10⁴ M⁻¹ greater or at least 10⁵ M⁻¹ greaterthan a binding constant for other molecules in a biological sample). Theterm “antibody” also includes genetically engineered forms such aschimeric antibodies (for example, humanized murine antibodies),heteroconjugate antibodies (such as, bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, J., Immunology, 3′ Ed., W.H. Freeman & Co., New York, 1997.

In terms of antibody structure, an immunoglobulin has heavy (H) chainsand light (L) chains interconnected by disulfide bonds. There are twotypes of light chain, lambda (O) and kappa (κ). There are five mainheavy chain classes (or isotypes) which determine the functionalactivity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavyand light chain contains a constant region and a variable region, (theregions are also known as “domains”). In combination, the heavy and thelight chain variable regions specifically bind the antigen. Light andheavy chain variable regions contain a “framework” region interrupted bythree hypervariable regions, also called “complementarity-determiningregions” or “CDRs”. The extent of the framework region and CDRs havebeen defined (see, Kabat et al., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1991, which ishereby incorporated by reference). The Kabat database is now maintainedonline. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, largely adopts a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, framework regions act to forma scaffold that provides for positioning the CDRs in correct orientationby inter-chain, non-covalent interactions.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H)CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds EGFR will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may be specifically bound by the products of specifichumoral or cellular immunity, such as an antibody molecule or T-cellreceptor. Antigens can be any type of molecule including, for example,haptens, simple intermediary metabolites, sugars (e.g.,oligosaccharides), lipids, and hormones as well as macromolecules suchas complex carbohydrates (e.g., polysaccharides), phospholipids, andproteins. Common categories of antigens include, but are not limited to,viral antigens, bacterial antigens, fungal antigens, protozoa and otherparasitic antigens, tumor antigens, antigens involved in autoimmunedisease, allergy and graft rejection, toxins, and other miscellaneousantigens.

As used herein, the term “antigen binding domain” refers to any proteinor polypeptide domain that can specifically bind to an antigen target.

The term “chimeric antigen receptor” (CAR), as used herein, refers to afused protein comprising an extracellular domain capable of binding toan antigen, a transmembrane domain derived from a polypeptide differentfrom a polypeptide from which the extracellular domain is derived, andat least one intracellular domain. The “chimeric antigen receptor (CAR)”is sometimes called a “chimeric receptor”, a “T-body”, or a “chimericimmune receptor (CIR).” The “extracellular domain capable of binding toan antigen” means any oligopeptide or polypeptide that can bind to acertain antigen. The “intracellular domain” means any oligopeptide orpolypeptide known to function as a domain that transmits a signal tocause activation or inhibition of a biological process in a cell. The“transmembrane domain” means any oligopeptide or polypeptide known tospan the cell membrane and that can function to link the extracellularand signaling domains. A chimeric antigen receptor may optionallycomprise a “hinge domain” which serves as a linker between theextracellular and transmembrane domains. Non-limiting exemplarypolynucleotide sequences that encode for components of each domain aredisclosed herein, e.g.:

Hinge domain: IgG1 heavy chain hinge sequence:CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGTransmembrane domain: CD28 transmembran region:TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGIntracellular domain: 4-1BB co-stimulatory signaling region:AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG Intracellular domain: CD28 co-stimulatorysignaling region: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCIntracellular domain: CD3 zeta signaling region:AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGCCCCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATCTGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

Further embodiments of each exemplary domain component include otherproteins that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% sequenceidentity with the proteins encoded by the above disclosed nucleic acidsequences. Further, non-limiting examples of such domains are providedherein.

The acronym “EGFR” stands for Epidermal Growth Factor Receptor. EGFRalso is known as ErbB-1 and HER1. It is the cell surface receptors ofthe epidermal growth factor family of cell surface receptors. The term“EGFR” also refers to a specific protein fragment associated with thisname and any other molecules that have analogous biological functionthat share at least 70%, or alternatively at least 80% amino acidsequence identity, preferably 900%6 sequence identity, more preferablyat least 95% amino acid sequence identity with any isoform of EGFR, asdisclosed herein. Isoform 1 is the canonical sequence; thus, allpositional information that follows refers to the amino acid sequencedisclosed below.

EGFR Isoform 1, Uniprot P00533-1:MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCCPPLMLYNPTTYQMDVNPEGDYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEIKDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA

Binding sites include but are not limited to positions 745 and 855,active sites include but are not limited to position 837; and othersites of interest include but are not limited to position 1016. EGFRIsoform 2 (Uniprot P00533-2) has an FL to LS substitution at position404 to 405 and is missing the region from position 406 to 1210. EGFRIsoform 4 (Uniprot P00533-4) has a C to S substitution at position 628and is missing the region from position 629 to 1210. EGFR Isoform 3(Uniprot P00533-3) differs from positons 628 to 705 and is missing theregion from position 706 to 1210, in accordance with the sequence below.

EGFR Isoform 3, Uniprot P00533-3:MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGPGNESLKAMLFCLFKLSSCNQSNDGSVSHQSGSPAAQESCLGWIPSLLPSEFQLGWGGC SHLHAWPSASVIITASSCH

EGFRvIII is a mutant form of EGFR that is reported to be expressed in aconsiderable proportion of patients with glioblastoma multiforme (GB).Gan et al. 205350-5370 report that the mutant form is expressed in othertumors as well. The term “mutant EGFR” may refer to EGFRvIII or aspecific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% aminoacid sequence identity with the EGFRvIII, as shown herein or anequivalent thereof as further defined herein.

EGFRvIII, Uniprot P00533[30-297]:MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQLGTFEDHFLS LQRMFNNCEV VLGNLEITYV QRNYDLSFLKTIQEVAGYVL IALNTVERIPLENLQIIRGN MYYENSYALAVLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVESIQWRDIVSS DFLSNMSMDF QNHLGSCQKCDPSCPNGSCWGAGEENCQKL TKIICAQQCS GRCRGKSPSDCCHNQCAAGC TGPRESDCLV CRKFRDEATC KDTCPPLMLYNPTTYQMDVN PEGKYSFGAT CVKKCPRNYV VTDHGSCVRACGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLSINATNIKHFK NCTSISGDLH ILPVAFRGDS FTHTPPLDPQELDILKTVKE ITGFLLIQAW PENRTDLHAF ENLEIIRGRTKQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLCYANTINWKKL FGTSGQKTKI ISNRGENSCK ATGQVCHALCSPEGCWGPEP RDCVSCRNVS RGRECVDKCN LLEGEPREFVENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPHCVKTCPAGVM GENNTLVWKY ADAGHVCHLC HPNCTYGCTGPGLEGCPTNG PKIPSIATGM VGALLLLLVV ALGIGLFMRRRHIVRKRTLR RLLQERELVE PLTPSGEAPN QATTRILKETEFKKIKVLGS GAFGTVYKGL WIPEGEKVKIPVAIKELREATSPKANKEIL DEAYVMASVD NPHVCRLLGICLTSTVQLIT QLMPFGCLLDYVREHKDNIG SQYLLNWCVQIAKGMNYLED RRLVHRDLAA RNVLVKTPQHVKITDFGLAKLLGAEEKEYH AEGGKVPIKW MALESILHRIYTHQSDVWSYGVTVWELMTF GSKPYDGIPA SEISSILEKGERLPQPPICT IDVYMIMVKC WMIDADSRPK FRELIIEFSKMARDPQRYLV IQGDERMHLPLMDEEDMDDV VDADEYLIPQQGFFSSPSTS RTPLLSSLSA TSNNSTVACIDRNGLQSCPIKEDSFLQRYS SDPTGALTED SIDDTFLPVPEYINQSVPKRPAGSVQNPVY HNQPLNPAPS RDPHYQDPHSTAVGNPEYLN TVQPTCVNSTFDSPAHWAQK GSHQISLDNPDYQQDFFPKE AKPNGIFKGS TAENAEYLRVAPQSSEFIGA

The term “mutant EGFR” may also refer to a natural variant of anyisoform of EGFR including but not limited variants with one or more ofthe following mutations: R to Q at position 98, P to R at position 266,G to D at position 428, R to K at position 521, V to I a position 674, Eto A at position 709, E to G at position 709, E to K at position 709, Gto A at position 719, G to C at position 719, G to D at position 719, Gto S at position 719, G to S at position 724, E to K at position 734,ELREATS to D at positions 746 to 752, ELREAT to A at positions 746 to751, a deletion from positions 746 to 750, a deletion at position 746, adeletion from positions 747 to 751, a deletion from positions 747 to749, L to F at position 747, R to P at position 748, a deletion frompositions 752 to 759, S to I at position 768, V to M at position 769, Qto R at position 787, T to M at position 790, L to V at position 833, Vto L at position 834, H to L at position 835, L to V at position 838, Lto M at position 858, L to R at position 858, L to Q at position 861, Gto E at position 873, R to G at position 962, H to P at position 988, Lto R at position 1034, A to V at position 1210, and/or a different aminoacid substitution or deletion at any one of the specific positions.

A “composition” typically intends a combination of the active agent,e.g., compound or composition, and a naturally-occurring ornon-naturally-occurring carrier, inert (for example, a detectable agentor label) or active, such as an adjuvant, diluent, binder, stabilizer,buffers, salts, lipophilic solvents, preservative, adjuvant or the likeand include pharmaceutically acceptable carriers. Carriers also includepharmaceutical excipients and additives proteins, peptides, amino acids,lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-,tri-, tetra-oligosaccharides, and oligosaccharides; derivatized sugarssuch as alditols, aldonic acids, esterified sugars and the like; andpolysaccharides or sugar polymers), which can be present singly or incombination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, arginine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like. Carbohydrate excipients are alsointended within the scope of this technology, examples of which includebut are not limited to monosaccharides such as fructose, maltose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, sucrose, trehalose, cellobiose, and the like;polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,starches, and the like; and alditols, such as mannitol, xylitol,maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

The term “consensus sequence” as used herein refers to an amino acid ornucleic acid sequence that is determined by aligning a series ofmultiple sequences and that defines an idealized sequence thatrepresents the predominant choice of amino acid or base at eachcorresponding position of the multiple sequences. Depending on thesequences of the series of multiple sequences, the consensus sequencefor the series can differ from each of the sequences by zero, one, afew, or more substitutions. Also, depending on the sequences of theseries of multiple sequences, more than one consensus sequence may bedetermined for the series. The generation of consensus sequences hasbeen subjected to intensive mathematical analysis. Various softwareprograms can be used to determine a consensus sequence.

As used herein, the term “CD8 α hinge domain” refers to a specificprotein fragment associated with this name and any other molecules thathave analogous biological function that share at least 70%, oralternatively at least 80% amino acid sequence identity, preferably 90%sequence identity, more preferably at least 95% sequence identity withthe CD8 α hinge domain sequence as shown herein. The example sequencesof CD8 α hinge domain for human, mouse, and other species are providedin Pinto. R. D. et al. (2006) Vet. Immunol. Immunopathol. 110:169-177.The sequences associated with the CD8 α hinge domain are provided inPinto, R. D. et al. (2006) Vet. Immunol. Immunopathol. 110:169-177.Non-limiting examples of such include:

Human CD8 alpha hinge domain,PAKPTTTPAPRPPTPAPTIASQPLSRPEACRPAAGGAVHTRGLDFACDIYMouse CD8 alpha hinge domain,KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYCat CD8 alpha hinge domain,PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY

As used herein, the term “CD8 α transmembrane domain” refers to aspecific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% sequenceidentity with the CD8 α transmembrane domain sequence as shown herein.The fragment sequences associated with the amino acid positions 183 to203 of the human T-cell surface glycoprotein CD8 alpha chain (NCBIReference Sequence: NP_001759.3), or the amino acid positions 197 to 217of the mouse T-cell surface glycoprotein CD8 alpha chain (NCBI ReferenceSequence: NP_001074579.1), and the amino acid positions 190 to 210 ofthe rat T-cell surface glycoprotein CD8 alpha chain (NCBI ReferenceSequence: NP_113726.1) provide additional example sequences of the CD8 αtransmembrane domain. The sequences associated with each of the listedNCBI are provided as follows:

Human CD8 alpha transmembrane domain: IYIWAPLAGTCGVLLLSLVITMouse CD8 alpha transmembrane domain: IWAPLAGICVALLLSLIITLIRat CD8 alpha transmembrane domain: IWAPLAGICAVLLLSLVITLI

As used herein, the term “4-1BB costimulatory signaling region” refersto a specific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% sequenceidentity with the 4-1BB costimulatory signaling region sequence as shownherein. The example sequences of the 4-1BB costimulatory signalingregion are provided in U.S. Publication 20130266551A1 (filed as U.S.application Ser. No. 13/826,258). The sequence of the 4-1BBcostimulatory signaling region associated disclosed in the U.S.application Ser. No. 13/826,258 is disclosed as follows:

The 4-1BB Costimulatory Signaling Region:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

As used herein, the term “CD28 transmembrane domain” refers to aspecific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity, atleast 90% sequence identity, or alternatively at least 95% sequenceidentity with the CD28 transmembrane domain sequence as shown herein.The fragment sequences associated with the GenBank Accession Nos:XM_006712862.2 and XM_009444056.1 provide additional, non-limiting,example sequences of the CD28 transmembrane domain. The sequencesassociated with each of the listed accession numbers are provided asfollows the sequence mentioned herein above.

As used herein, the term “CD28 costimulatory signaling region” refers toa specific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% sequenceidentity with the CD28 costimulatory signaling region sequence shownherein. The CD28 costimulatory region comprises an transmembrane domainand an intracellular domain. The example sequences CD28 costimulatorysignaling domain are provided in U.S. Pat. No. 5,686,281; Geiger, T. L.et al., Blood 98: 2364-2371 (2001); Hombach, A. et al., J Immunol 167:6123-6131 (2001); Maher, J. et al. Nat Biotechnol 20: 70-75 (2002);Haynes, N. M. et al., J Immunol 169: 5780-5786 (2002); Haynes, N. M. etal., Blood 100: 3155-3163 (2002). Non-limiting examples include residues114-220 of the below CD28 Sequence:

MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSCKYSYNLFSRE FRASLHKGLDSAVECVVYG NYSQQLQVYSKTGFNCDGKL GNESVTFYLQ YYVNQTDIYFCKIEVMYPPPYLDNEKSNG TIIHVKGKHL CPSFLFPGPSKPFWVLVVVG GVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS,and equivalents thereof.

As used herein, the term “ICOS costimulatory signaling region” refers toa specific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% sequenceidentity with the ICOS costimulatory signaling region sequence as shownherein. Non-limiting example sequences of the ICOS costimulatorysignaling region are provided in U.S. Publication 2015/0017141 A1 theexemplary polynucleotide sequence provided below.

ICOS Costimulatory Signaling Region:

ACAAAAAAGA AGTATTCATC CAGTGTGCAC GACCCTAACGGTGAATACAT GTTCATGAGA GCAGTGAACA CAGCCAAAAA ATCCAGACTC ACAGATGTGA CCCTA

As used herein, the term “OX40 costimulatory signaling region” refers toa specific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity, oralternatively 90% sequence identity, or alternatively at least 95%sequence identity with the OX40 costimulatory signaling region sequenceas shown herein. Non-limiting example sequences of the OX40costimulatory signaling region are disclosed in U.S. Publication2012/20148552A 1, and include the exemplary sequence provided below.

OX40 Costimulatory Signaling Region, SEQ ID NO:72:

AGGGACCAG AGGCTGCCCC CCGATGCCCA CAAGCCCCCTGGGGGAGGCA GTTTCCGGAC CCCCATCCAA GAGGAGCAGGCCGACGCCCA CTCCACCCTG GCCAAGATC

As used herein, the term “CD3 zeta signaling domain” refers to aspecific protein fragment associated with this name and any othermolecules that have analogous biological function that share at least70%, or alternatively at least 80% amino acid sequence identity,preferably 90% sequence identity, more preferably at least 95% sequenceidentity with the CD3 zeta signaling domain sequence as shown herein.The example sequences of the CD3 zeta signaling domain are provided inU.S. application Ser. No. 13/826,258. The sequence associated with theCD3 zeta signaling domain is listed as follows:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR

As used herein, the term “B cell,” refers to a type of lymphocyte in thehumoral immunity of the adaptive immune system. B cells principallyfunction to make antibodies, serve as antigen presenting cells, releasecytokines, and develop memory B cells after activation by antigeninteraction. B cells are distinguished from other lymphocytes, such as Tcells, by the presence of a B-cell receptor on the cell surface. B cellsmay either be isolated or obtained from a commercially available source.Non-limiting examples of commercially available B cell lines includelines AHH-1 (ATCC® CRL-8146T), BC-1 (ATCC® CRL-2230™), BC-2 (ATCC®CRL-2231™), BC-3 (ATCC® CRL-2277™), CA46 (ATCC® CRL-1648™), DG-75[D.G.-75] (ATCC® CRL-2625™), DS-1 (ATCC® CRL-11102™), EB-3 [EB3] (ATCC®CCL-85™), Z-138 (ATCC #CRL-3001), DB (ATCC CRL-2289), Toledo (ATCCCRL-2631), Pfiffer (ATCC CRL-2632), SR (ATCC CRL-2262), JM-1 (ATCCCRL-10421), NFS-5 C-1 (ATCC CRL-1693); NFS-70 C10 (ATCC CRL-1694),NFS-25 C-3 (ATCC CRL-1695), AND SUP-B15 (ATCC CRL-1929). Furtherexamples include but are not limited to cell lines derived fromanaplastic and large cell lymphomas, e.g., DEL, DL-40, FE-PD, JB6,Karpas 299, Ki-JK, Mac-2A Ply1, SR-786, SU-DHL-1, -2, -4, -5, -6, -7,-8, -9, -10, and -16, DOHH-2, NU-DHL-1, U-937, Granda 519, USC-DHL-1,RL; Hodgkin's lymphomas, e.g., DEV, HD-70, HDLM-2, HD-MyZ, HKB-1, KM-H2,L 428, L 540, L1236, SBH-1, SUP-HD1, SU/RH-HD-1. Non-limiting exemplarysources for such commercially available cell lines include the AmericanType Culture Collection, or ATCC, (www.atcc.org/) and the GermanCollection of Microorganisms and Cell Cultures (https://www.dsmz.de/).

As used herein, the term “T cell,” refers to a type of lymphocyte thatmatures in the thymus. T cells play an important role in cell-mediatedimmunity and are distinguished from other lymphocytes, such as B cells,by the presence of a T-cell receptor on the cell surface. T-cells mayeither be isolated or obtained from a commercially available source. “Tcell” includes all types of immune cells expressing CD3 includingT-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), naturalkiller T-cells, T-regulatory cells (Treg) and gamma-delta T cells. A“cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, andneutrophils, which cells are capable of mediating cytotoxicityresponses. Non-limiting examples of commercially available T-cell linesinclude lines BCL2 (AAA) Jurkat (ATCC® CRL-2902™), BCL2 (S70A) Jurkat(ATCC® CRL-2900™), BCL2 (S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat(ATCC® CRL-2899™), Neo Jurkat (ATCC® CRL-2898™), TALL-104 cytotoxichuman T cell line (ATCC #CRL-11386). Further examples include but arenot limited to mature T-cell lines, e.g., such as Deglis, EBT-8,HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3,SMZ-1 and T34; and immature T-cell lines, e.g., ALL-SIL, Be13, CCRF-CEM,CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1,Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT3, MOLT-4, MOLT 13, MOLT-16, MT-1, MT-ALL, P12/Ichikawa, Peer, PERO117,PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1,TALL-101, TALL-103/2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197,TK-6, TLBR-1, -2, -3, and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCCTIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4; 11(ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and cutaneous T-celllymphoma lines, e.g., HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294),HuT102 (ATCC TIB-162). Null leukemia cell lines, including but notlimited to REH, NALL-1, KM-3, L92-221, are a another commerciallyavailable source of immune cells, as are cell lines derived from otherleukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocyticleukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1leukemia, KG-1 leukemia, U266 myeloma. Non-limiting exemplary sourcesfor such commercially available cell lines include the American TypeCulture Collection, or ATCC, (http://www.atcc.org/) and the GermanCollection of Microorganisms and Cell Cultures (https://www.dsmz.de/).

As used herein, the term “NK cell,” also known as natural killer cell,refers to a type of lymphocyte that originates in the bone marrow andplay a critical role in the innate immune system. NK cells provide rapidimmune responses against viral-infected cells, tumor cells or otherstressed cell, even in the absence of antibodies and majorhistocompatibility complex on the cell surfaces. NK-92 (ATCC, CRL-2407)and NKL (described in Robertson et al. (1996) 24(3):406-414) arespecific examples of NK cells. NK cells may either be isolated orobtained from a commercially available source. Non-limiting examples ofcommercial NK cell lines include lines NK-92 (ATCC® CRL-2407™), NK-92MI(ATCC® CRL-2408™). Further examples include but are not limited to NKlines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplarysources for such commercially available cell lines include the AmericanType Culture Collection, or ATCC, (http://www.atcc.org/) and the GermanCollection of Microorganisms and Cell Cultures (https://www.dsmz.de/).

As used herein, the terms “nucleic acid sequence” and “polynucleotide”are used interchangeably to refer to a polymeric form of nucleotides ofany length, either ribonucleotides or deoxyribonucleotides. Thus, thisterm includes, but is not limited to, single-, double-, ormulti-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or apolymer comprising purine and pyrimidine bases or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases.

The term “encode” as it is applied to nucleic acid sequences refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, the term signal peptide or signal polypeptide intends anamino acid sequence usually present at the N-terminal end of newlysynthesized secretory or membrane polypeptides or proteins. It acts todirect the polypeptide across or into a cell membrane and is thensubsequently removed. Examples of such are well known in the art.Non-limiting examples are those described in U.S. Pat. Nos. 8,853,381and 5,958,736.

As used herein, the term “vector” refers to a nucleic acid constructdeigned for transfer between different hosts, including but not limitedto a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc. In someembodiments, plasmid vectors may be prepared from commercially availablevectors. In other embodiments, viral vectors may be produced frombaculoviruses, retroviruses, adenoviruses, AAVs, etc. according totechniques known in the art. In one embodiment, the viral vector is alentiviral vector.

The term “promoter” as used herein refers to any sequence that regulatesthe expression of a coding sequence, such as a gene. Promoters may beconstitutive, inducible, repressible, or tissue-specific, for example. A“promoter” is a control sequence that is a region of a polynucleotidesequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors.

As used herein, the term “isolated cell” generally refers to a cell thatis substantially separated from other cells of a tissue. “Immune cells”includes, e.g., white blood cells (leukocytes) which are derived fromhematopoietic stem cells (HSC) produced in the bone marrow, lymphocytes(T cells, B cells, natural killer (NK) cells) and myeloid-derived cells(neutrophil, eosinophil, basophil, monocyte, macrophage, dendriticcells). “T cell” includes all types of immune cells expressing CD3including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells),natural killer T-cells, T-regulatory cells (Treg) and gamma-delta Tcells. A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK)cells, and neutrophils, which cells are capable of mediatingcytotoxicity responses.

As used herein, the term “isolated cell” generally refers to a cell thatis substantially separated from other cells of a tissue. The termincludes prokaryotic and eukaryotic cells.

“Immune cells” includes, e.g., white blood cells (leukocytes) which arederived from hematopoietic stem cells (HSC) produced in the bone marrow,lymphocytes (T cells, B cells, natural killer (NK) cells) andmyeloid-derived cells (neutrophil, eosinophil, basophil, monocyte,macrophage, dendritic cells). “T cell” includes all types of immunecells expressing CD3 including T-helper cells (CD4+ cells), cytotoxicT-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg)and gamma-delta T cells. A “cytotoxic cell” includes CD8+ T cells,natural-killer (NK) cells, and neutrophils, which cells are capable ofmediating cytotoxicity responses.

The term “transduce” or “transduction” as it is applied to theproduction of chimeric antigen receptor cells refers to the processwhereby a foreign nucleotide sequence is introduced into a cell. In someembodiments, this transduction is done via a vector.

As used herein, the term “autologous,” in reference to cells refers tocells that are isolated and infused back into the same subject(recipient or host). “Allogeneic” refers to non-autologous cells.

An “effective amount” or “efficacious amount” refers to the amount of anagent, or combined amounts of two or more agents, that, whenadministered for the treatment of a mammal or other subject, issufficient to effect such treatment for the disease. The “effectiveamount” will vary depending on the agent(s), the disease and itsseverity and the age, weight, etc., of the subject to be treated.

As used herein, the term “cancer” refers to any one of a group ofdiseases involving abnormal cell growth with the potential to invade orspread to other parts of the body. Cancer can affect different parts ofthe body of a subject. Non-limiting examples of cancer include breastcancer, ovarian cancer, leukemia, lymphoma, glioblastoma, andastrocytoma. In certain embodiments, cancers originating in the brainand/or metastases of cancers from other parts of the body that occur inthe brain are of relevance to this disclosure. For example,“glioblastoma” (GB) is a tumor arising from astrocytes that is highlymalignant; it is suspected that this is because of rapid reproduction ofcells and angiogenesis. A “solid tumor” is an abnormal mass of tissuethat usually does not contain cysts or liquid areas. Solid tumors can bebenign or malignant. Different types of solid tumors are named for thetype of cells that form them. Examples of solid tumors include sarcomas,carcinomas, and lymphomas.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Forexample, a composition consisting essentially of the elements as definedherein would not exclude trace contaminants from the isolation andpurification method and pharmaceutically acceptable carriers, such asphosphate buffered saline, preservatives and the like. “Consisting of”shall mean excluding more than trace elements of other ingredients andsubstantial method steps for administering the compositions disclosedherein. Aspects defined by each of these transition terms are within thescope of the present disclosure.

As used herein, the term “detectable marker” refers to at least onemarker capable of directly or indirectly, producing a detectable signal.A non-exhaustive list of this marker includes enzymes which produce adetectable signal, for example by colorimetry, fluorescence,luminescence, such as horseradish peroxidase, alkaline phosphatase,l-galactosidase, glucose-6-phosphate dehydrogenase, chromophores such asfluorescent, luminescent dyes, groups with electron density detected byelectron microscopy or by their electrical property such asconductivity, amperometry, voltammetry, impedance, detectable groups,for example whose molecules are of sufficient size to induce detectablemodifications in their physical and/or chemical properties, suchdetection may be accomplished by optical methods such as diffraction,surface plasmon resonance, surface variation, the contact angle changeor physical methods such as atomic force spectroscopy, tunnel effect, orradioactive molecules such as ³²P, ³⁵S or ¹²⁵I.

As used herein, the term “purification marker” refers to at least onemarker useful for purification or identification. A non-exhaustive listof this marker includes His, lacZ, GST, maltose-binding protein, NusA,BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry, thioredoxin, poly(NANP), V5,Snap, HA, chitin-binding protein, Softag 1, Softag 3, Strep, orS-protein. Suitable direct or indirect fluorescence marker compriseFLAG, GFP, YFP, RFP, dTomato, cherry, Cy3, Cy 5, Cy 5.5, Cy 7, DNP,AMCA, Biotin, Digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors,FITC, TRITC or any other fluorescent dye or hapten.

As used herein, the term “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in a eukaryotic cell.The expression level of a gene may be determined by measuring the amountof mRNA or protein in a cell or tissue sample. In one aspect, theexpression level of a gene from one sample may be directly compared tothe expression level of that gene from a control or reference sample. Inanother aspect, the expression level of a gene from one sample may bedirectly compared to the expression level of that gene from the samesample following administration of a compound.

As used herein, “homology” or “identical”, percent “identity” or“similarity”, when used in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences or subsequencesthat are the same or have a specified percentage of nucleotides or aminoacid residues that are the same, e.g., at least 60% identity, preferablyat least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or higher identity over a specified region (e.g.,nucleotide sequence encoding an antibody described herein or amino acidsequence of an antibody described herein). Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. Thealignment and the percent homology or sequence identity can bedetermined using software programs known in the art, for example thosedescribed in Current Protocols in Molecular Biology (Ausubel et al.,eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably,default parameters are used for alignment. A preferred alignment programis BLAST, using default parameters. In particular, preferred programsare BLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.The terms “homology” or “identical”, percent “identity” or “similarity”also refer to, or can be applied to, the complement of a test sequence.The terms also include sequences that have deletions and/or additions,as well as those that have substitutions. As described herein, thepreferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is at least50-100 amino acids or nucleotides in length. An “unrelated” or“non-homologous” sequence shares less than 40% identity, oralternatively less than 25% identity, with one of the sequencesdisclosed herein.

The phrase “first line” or “second line” or “third line” refers to theorder of treatment received by a patient. First line therapy regimensare treatments given first, whereas second or third line therapy aregiven after the first line therapy or after the second line therapy,respectively. The National Cancer Institute defines first line therapyas “the first treatment for a disease or condition. In patients withcancer, primary treatment can be surgery, chemotherapy, radiationtherapy, or a combination of these therapies. First line therapy is alsoreferred to those skilled in the art as “primary therapy and primarytreatment.” See National Cancer Institute website at www.cancer.gov,last visited on May 1, 2008. Typically, a patient is given a subsequentchemotherapy regimen because the patient did not show a positiveclinical or sub-clinical response to the first line therapy or the firstline therapy has stopped.

In one aspect, the term “equivalent” or “biological equivalent” of anantibody or fragment thereof means the ability of the antibody toselectively bind its epitope protein or fragment thereof as measured byELISA or other suitable methods. Biologically equivalent antibodiesinclude, but are not limited to, those antibodies, peptides, antibodyfragments, antibody variant, antibody derivative and antibody mimeticsthat bind to the same epitope as the reference antibody.

It is to be inferred without explicit recitation and unless otherwiseintended, that when the present disclosure relates to a polypeptide,protein, polynucleotide or antibody, an equivalent or a biologicallyequivalent of such is intended within the scope of this disclosure. Asused herein, the term “biological equivalent thereof” is intended to besynonymous with “equivalent thereof” when referring to a referenceprotein, antibody, polypeptide or nucleic acid, intends those havingminimal homology while still maintaining desired structure orfunctionality. Unless specifically recited herein, it is contemplatedthat any polynucleotide, polypeptide or protein mentioned herein alsoincludes equivalents thereof. For example, an equivalent intends atleast about 70% homology or identity, or at least 80% homology oridentity and alternatively, or at least about 85%, or alternatively atleast about 90%, or alternatively at least about 95%, or alternatively98% percent homology or identity and exhibits substantially equivalentbiological activity to the reference protein, polypeptide or nucleicacid. Alternatively, when referring to polynucleotides, an equivalentthereof is a polynucleotide that hybridizes under stringent conditionsto the reference polynucleotide or its complement.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) having a certain percentage (for example, 80%, 85%,90° %, or 95%) of “sequence identity” to another sequence means that,when aligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. The alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in Current Protocols in MolecularBiology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table7.7.1. Preferably, default parameters are used for alignment. Apreferred alignment program is BLAST, using default parameters. Inparticular, preferred programs are BLASTN and BLASTP, using thefollowing default parameters: Genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10× SSC; formamide concentrationsof about 0% to about 25%; and wash solutions from about 4×SSC to about8×SSC Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1×SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It isunderstood that equivalents of SSC using other buffer systems can beemployed.

A “normal cell corresponding to the tumor tissue type” refers to anormal cell from a same tissue type as the tumor tissue. A non-limitingexample is a normal lung cell from a patient having lung tumor, or anormal colon cell from a patient having colon tumor.

The term “isolated” as used herein refers to molecules or biologicals orcellular materials being substantially free from other materials. In oneaspect, the term “isolated” refers to nucleic acid, such as DNA or RNA,or protein or polypeptide (e.g., an antibody or derivative thereof), orcell or cellular organelle, or tissue or organ, separated from otherDNAs or RNAs, or proteins or polypeptides, or cells or cellularorganelles, or tissues or organs, respectively, that are present in thenatural source. The term “isolated” also refers to a nucleic acid orpeptide that is substantially free of cellular material, viral material,or culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.The term “isolated” is also used herein to refer to cells or tissuesthat are isolated from other cells or tissues and is meant to encompassboth cultured and engineered cells or tissues.

As used herein, the term “monoclonal antibody” refers to an antibodyproduced by a single clone of B-lymphocytes or by a cell into which thelight and heavy chain genes of a single antibody have been transfected.Monoclonal antibodies are produced by methods known to those of skill inthe art, for instance by making hybrid antibody-forming cells from afusion of myeloma cells with immune spleen cells. Monoclonal antibodiesinclude humanized monoclonal antibodies.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another aspect, the subunit may belinked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any aspect of this technology that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiednucleic acid, peptide, protein, biological complexes or other activecompound is one that is isolated in whole or in part from proteins orother contaminants. Generally, substantially purified peptides,proteins, biological complexes, or other active compounds for use withinthe disclosure comprise more than 80% of all macromolecular speciespresent in a preparation prior to admixture or formulation of thepeptide, protein, biological complex or other active compound with apharmaceutical carrier, excipient, buffer, absorption enhancing agent,stabilizer, preservative, adjuvant or other co-ingredient in a completepharmaceutical formulation for therapeutic administration. Moretypically, the peptide, protein, biological complex or other activecompound is purified to represent greater than 90%, often greater than95% of all macromolecular species present in a purified preparationprior to admixture with other formulation ingredients. In other cases,the purified preparation may be essentially homogeneous, wherein othermacromolecular species are not detectable by conventional techniques.

As used herein, the term “specific binding” means the contact between anantibody and an antigen with a binding affinity of at least 10⁻⁶ M. Incertain aspects, antibodies bind with affinities of at least about 10⁻⁷M, and preferably 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰M, 10⁻¹¹ M, or 10⁻¹² M.

As used herein, the term “recombinant protein” refers to a polypeptidewhich is produced by recombinant DNA techniques, wherein generally, DNAencoding the polypeptide is inserted into a suitable expression vectorwhich is in turn used to transform a host cell to produce theheterologous protein.

As used herein, “treating” or “treatment” of a disease in a subjectrefers to (1) preventing the symptoms or disease from occurring in asubject that is predisposed or does not yet display symptoms of thedisease; (2) inhibiting the disease or arresting its development; or (3)ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, “treatment” is an approach forobtaining beneficial or desired results, including clinical results. Forthe purposes of the present technology, beneficial or desired resultscan include one or more, but are not limited to, alleviation oramelioration of one or more symptoms, diminishment of extent of acondition (including a disease), stabilized (i.e., not worsening) stateof a condition (including disease), delay or slowing of condition(including disease), progression, amelioration or palliation of thecondition (including disease), states and remission (whether partial ortotal), whether detectable or undetectable.

As used herein, the term “overexpress” with respect to a cell, a tissue,or an organ expresses a protein to an amount that is greater than theamount that is produced in a control cell, a control issue, or an organ.A protein that is overexpressed may be endogenous to the host cell orexogenous to the host cell.

As used herein the term “linker sequence” relates to any amino acidsequence comprising from 1 to 10, or alternatively, 8 amino acids, oralternatively 6 amino acids, or alternatively 5 amino acids that may berepeated from 1 to 10, or alternatively to about 8, or alternatively toabout 6, or alternatively about 5, or 4 or alternatively 3, oralternatively 2 times. Non-limiting examples of linker sequences areknown in the art, e.g., GGGGSGGGGSGGGG; the tripeptide EFM; orGlu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met

As used herein, the term “enhancer”, as used herein, denotes sequenceelements that augment, improve or ameliorate transcription of a nucleicacid sequence irrespective of its location and orientation in relationto the nucleic acid sequence to be expressed. An enhancer may enhancetranscription from a single promoter or simultaneously from more thanone promoter. As long as this functionality of improving transcriptionis retained or substantially retained (e.g., at least 70%, at least 80%,at least 90% or at least 95% of wild-type activity, that is, activity ofa full-length sequence), any truncated, mutated or otherwise modifiedvariants of a wild-type enhancer sequence are also within the abovedefinition.

As used herein, the term “WPRE” or “Woodchuck Hepatitis Virus (WHP)Post-transcriptional Regulatory Element” refers to a specific nucleotidefragment associated with this name and any other molecules that haveanalogous biological function that share at least 70%, or alternativelyat least 80% amino acid sequence identity, preferably 90% sequenceidentity, more preferably at least 95% sequence identity with the WPREsequence as shown herein. For example, WPRE refers to a region similarto the human hepatitis B virus posttranscriptional regulatory element(HBVPRE) present in the Woodchuck hepatitis virus genomic sequence(GenBank Accession No. J04514), and that the 592 nucleotides fromposition 1093 to 1684 of this genomic sequence correspond to thepost-transcriptional regulatory region (Journal of Virology, Vol. 72, p.5085-5092, 1998). The analysis using retroviral vectors revealed thatWPRE inserted into the 3′-terminal untranslated region of a gene ofinterest increases the amount of protein produced by 5 to 8 folds. Ithas also been reported that the introduction of WPRE suppresses mRNAdegradation (Journal of Virology, Vol. 73, p. 2886-2892, 1999). In abroad sense, elements such as WPRE that increase the efficiency of aminoacid translation by stabilizing mRNAs are also thought to be enhancers.

The term “oncolytic” as used in reference to a virus describes a virusthat preferentially infects and kills cancer cells. A “herpes virus”refers to any member of the family herpesviridae, including but notlimited to herpes simplex viruses (like HSV-1 and HSV-2). Non-limitingexemplary oncolytic herpes viruses and use thereof are described herein.For example, G207, HSV1716, NV1020, Talimogene laherparvec (“T-VEC” or“Oncovex-GMCSF”) have all been tested in clinical trials. See Vargheseet al. (2002) Cancer Gene Therapy. 9(12):967-978.

LIST OF ABBREVIATIONS

CAR: chimeric antigen receptorIRES: internal ribosomal entry siteMFI: mean fluorescence intensityMOI: multiplicity of infectionPBMC: peripheral blood mononuclear cellsPBS: phosphate buffered salinescFv: single chain variable fragmentGB: glioblastomaBCBM: breast cancer brain metastasisoHSV: oncolytic herpes simplex virus

MODES FOR CARRYING OUT THE DISCLOSURE Chimeric Antigen Receptors andUses Thereof

Compositions

The present disclosure provides chimeric antigen receptors (CAR) thatbind to wild-type and/or mutant EGFR comprising, or consistingessentially of, an extracellular and intracellular domain. Theextracellular domain comprises a target-specific binding elementotherwise referred to as the antigen binding domain. The intracellulardomain or cytoplasmic domain comprises a costimulatory signaling regionand a zeta chain portion. The CAR may optionally further comprise aspacer domain of up to 300 amino acids, preferably 10 to 100 aminoacids, more preferably 25 to 50 amino acids. In one aspect, the presentdisclosure provides a chimeric antigen receptor (CAR) that comprises, oralternatively consists essentially of, or yet further consists of: (a)an antigen binding domain of an anti-EGFR antibody that recognizeseither or both wild type and/or mutant Epidermal Growth Factor Receptor(EGFR) (“wt EGFR and/or mutant EGFR”); (b) a hinge domain polypeptide;(c) a costimulatory molecule or polypeptide; and (d) a CD3 zetasignaling domain.

Antigen Binding Domain.

In certain aspects, the present disclosure provides a CAR thatcomprises, or alternatively consists essentially thereof, or yetconsists of, an antigen binding domain specific to wild-type and/ormutant EGFR. In one aspect the mutant EGFR is EGFRvIII. In someembodiments, the antigen binding domain comprises, or alternativelyconsists essentially thereof, or yet consists of the antigen bindingdomain of a wt and/or mutant EGFR antibody. In further embodiments, theantigen binding domain comprises, or alternatively consists essentiallyof, or yet further consists of, the heavy chain variable region andlight chain variable region of an anti-EGFR antibody that in turn,comprises, or alternatively consists essentially thereof, or yetconsists of, the antigen binding domain the anti-EGFR antibody. Lightchain and heavy chain variable regions for anti-EGFR antibodies areknown in the art and described above.

In some embodiments, the heavy chain variable region of the antibodycomprises, or consists essentially thereof, or consists of:

Q V Q L Q Q S G S E M A R P G A S V K L P C K A S G D T F T S Y W M H WV K Q R H G H G P E W I G N I Y P G S G G T N Y A E K F K N K V T L T VD R S S R T V Y M H L S R L T S E D S A V Y Y C T R S G G P Y F F D Y WG Q G T T L T V S S, or an equivalent thereof, or a polynucleotideencoded by the polypeptide:GACATTCTAATGACCCAATCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCTACCTGCAAAGGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCGACCGATTTTACCTGCAAAGGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCGACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTAGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATATTCCTCCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTGCGGCC, or an equivalent thereof.

The polypeptide or equivalents of each thereof, can be followed by anadditional 50 amino acids, or alternatively about 40 amino acids, oralternatively about 30 amino acids, or alternatively about 20 aminoacids, or alternatively about 10 amino acids, or alternatively about 5amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids atthe carboxy-terminus.

In other aspect, the LC variable region comprises, or alternativelyconsists essentially of, or yet further consists of:

D I L M T Q S P L S L P V S L G D Q A S I S C R S S Q N I V H N N G I TY L E W Y L Q R P G Q S P K L L I Y K V S D R F S G V P D R F S G S G SG T D F T L K I S R V E A E D L G I Y Y C F Q G S H I P P T F G G G T KL E I K R A A, or an equivalent thereof, or a polypeptide encoded by thepolynucleotide:CAGGTCCAGCTGCAGCAGTCTGGGTCTGAGATGGCGAGGCCTGGAGCTTCAGTGAAGCTGCCCTGCAAGGCTTCTGGCGACACATTCACCAGTTACTGGATGCACTGGGTGAAGCAGAGGCATGGACATGGCCCTGAGTGGATCGGAAATATTTATCCAGGTATGAAGTACTAACTACGCTGAGAAGTTCAAGAACAAGGTCACTCTGACTGTAGACAGGTCCTCCCGCACAGTCTACATGCACCTCAGCAGGCTGACATCTGAGGACTCTGCGGTCTATTATTGTACAAGATCGGGGGGTCCCTACTTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCC, or an equivalent thereof.

The polypeptide or equivalents of each thereof, can be followed by anadditional 50 amino acids, or alternatively about 40 amino acids, oralternatively about 30 amino acids, or alternatively about 20 aminoacids, or alternatively about 10 amino acids, or alternatively about 5amino acids, or alternatively about 4, or 3, or 2 or 1 amino acids atthe carboxy-terminus.

An equivalent thereof comprises an polypeptide having at least 80% aminoacid identity to the CAR or a polypeptide that is encoded by apolynucleotide that hybridizes under conditions of high stringency tothe complement of a polynucleotide encoding the CAR, wherein conditionsof high stringency comprises incubation temperatures of about 55° C. toabout 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC;formamide concentrations of about 55% to about 75%; and wash solutionsof about 1×SSC, 0.1×SSC, or deionized water.

Alternative embodiments include one or more of the CDRs (e.g., CDR1,CDR2, CDR3) from the LC variable region with appropriate CDRs from otherEGFR antibody CDRs. And equivalents of each thereof. Accordingly, and asan example, the CDR1 and CDR2 from the LC variable region can becombined with the CDR3 of another anti-EGFR antibody's LC variableregion, and in some aspects, can include an additional 50 amino acids,or alternatively about 40 amino acids, or alternatively about 30 aminoacids, or alternatively about 20 amino acids, or alternatively about 10amino acids, or alternatively about 5 amino acids, or alternativelyabout 4, or 3, or 2 or 1 amino acids at the carboxy-terminus.

Transmembrane Domain.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from CD8, CD28, CD3,CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154, TCR. Alternatively the transmembrane domain may besynthetic, in which case it will comprise predominantly hydrophobicresidues such as leucine and valine. Preferably a triplet ofphenylalanine, tryptophan and valine will be found at each end of asynthetic transmembrane domain. Optionally, a short oligo- orpolypeptide linker, preferably between 2 and 10 amino acids in lengthmay form the linkage between the transmembrane domain and thecytoplasmic signaling domain of the CAR. A glycine-serine doubletprovides a particularly suitable linker. In one aspect, thetransmembrane domain is a CD28 transmembrane domain, non-limitingexamples of such are described above. In another aspect, thetransmembrane domain is a CD8a transmembrane domain that is linked to aCD8a hinge domain.

Cytoplasmic Domain.

The cytoplasmic domain or intracellular signaling domain of the CAR isresponsible for activation of at least one of the traditional effectorfunctions of an immune cell in which a CAR has been placed. Theintracellular signaling domain refers to a portion of a protein whichtransduces the effector function signal and directs the immune cell toperform its specific function. An entire signaling domain or a truncatedportion thereof may be used so long as the truncated portion issufficient to transduce the effector function signal. Cytoplasmicsequences of the TCR and co-receptors as well as derivatives or variantsthereof can function as intracellular signaling domains for use in aCAR. Intracellular signaling domains of particular use in this inventionmay be derived from CD8a, CD28, FcR, TCR, CD3, CDS, CD22, CD79a, CD79b,CD66d. Since signals generated through the TCR are alone insufficientfor full activation of a T cell, a secondary or co-stimulatory signalmay also be required. Thus, the intracellular region of a co-stimulatorysignaling molecule, including but not limited to CD8α, CD27, CD28, 4-IBB(CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand thatspecifically binds with CD83, to may also be included in the cytoplasmicdomain of the CAR. In one aspect, the intracellular domain is a CD28intracellular domain. In another aspect, it is a CD8a intracellulardomain.

In some embodiments, the cell activation moiety of the chimeric antigenreceptor is a T-cell signaling domain comprising, or alternativelyconsisting essentially of, or yet further consisting of, one or moreproteins or fragments thereof selected from the group consisting of CD8protein, CD28 protein, 4-1BB protein, and CD3-zeta protein.

In specific embodiments, the CAR comprises, or alternatively consistsessentially thereof, or yet consists of an antigen binding domain of anantibody that binds EGFR and mutant EGFR, a hinge domain, acostimulatory signaling region, and a CD3 zeta signaling domain. Infurther embodiments, the costimulatory signaling region comprises eitheror both a CD28 costimulatory signaling region and/or a 4-IBBcostimulatory signaling region. The CAR can further comprise a signalpolypeptide.

In some embodiments, the CAR can further comprise a detectable marker orpurification marker.

Also provided herein is an isolated complex comprising the EGFR CAR andan EGFR protein or a fragment thereof and/or a mutant EGFR protein or afragment thereof. Yet further provided is an isolated complex comprisingthe CAR as described herein a cell expressing wt EGFR and/or mutantEGFR.

Polynucleotides and Host Cells

This disclosure also provide isolated nucleic acids encoding the EGFRCAR as described above and the complements of these sequences. Thenucleic acids can be DNA, RNA or modified nucleic acids. The nucleicacids can be incorporated into a vector, e.g., a plasmid or viralvector, e.g., a lentiviral vector, an adenoviral vector, or anadeno-associated viral vector. In one aspect the vector is the pCDHvector.

This disclosure also provides isolated host cells (prokaryotic oreukaryotic) cells comprising an EGFR CAR as described herein or apolynucleotide encoding the CAR or their complements or vectorscomprising the polynucleotides or their complements. Non-limitingexamples of prokaryotic cells include a bacteria, e.g., E. coli.Non-limiting examples of eukaryotic cells include, T cells, NK cells,stem cells, 293 T cells NK-92 cells and NKL cells. The cells can be fromany species, e.g., an animal, a mammal, a human, a murine, an equine, abovine, a feline or a canine. The cells can be isolated from the patientto be treated. They therefore can be autologous or allogeneic. Thetransformed isolated cells can be used diagnostically ortherapeutically.

The isolated nucleic acids can further comprise a detectable marker or apurification marker. The nucleic acids can be combined with a carrier,e.g., a solid support or a pharmaceutically acceptable carrier. They areuseful to prepare the CARs as described herein.

Process for Preparing CARS

Aspects of the present disclosure relate to an isolated cell comprisingthe EGFR CAR and methods of producing such cells. The cell is aprokaryotic or a eukaryotic cell. In one aspect, the cell is a T cell oran NK cell. The eukaryotic cell can be from any preferred species, e.g.,an animal cell, a mammalian cell such as a human, an equine, a feline ora canine cell.

In specific embodiments, the isolated cell comprises, or alternativelyconsists essentially of, or yet further consists of an exogenous CARcomprising, or alternatively consisting essentially of, or yet furtherconsisting of, an antigen binding domain of an antibody thatspecifically recognized wildtype EGFR and/or mutant EGFR, a hingedomain, a costimulatory molecule (e.g. a CD28 costimulatory signalingregion and/or a 4-1BB costimulatory signaling region), and a CD3 zetasignaling domain. In certain embodiments, the isolated cell is a T-cell,e.g., an animal T-cell, a mammalian T-cell, a feline T-cell, a canineT-cell or a human T-cell. In certain embodiments, the isolated cell isan NK-cell, e.g., an animal NK-cell, a mammalian NK-cell, a felineNK-cell, a canine NK-cell or a human NK-cell.

In certain embodiments, methods of producing EGFR CAR expressing cellsare disclosed comprising, or alternatively consisting essentially of:(i) transducing a cell or a population of isolated cells with a nucleicacid sequence encoding the anti-EGFR CAR. The cell can be transducedusing the viral vectors as described herein or using technologydescribed in Riet, et al. (2103) Meth. Mol. Biol. 969:187-201 entitled“Nonviral RNA transfection to transiently modify T cell with chimericantigen receptors for adoptive therapy.” In a further aspect, the methodfurther comprises selecting for a cell that expresses the CAR byselected cells that express the EGFR binding domain Selection can beaccomplished by use of anti-EGFR antibodies to selectively recognize andbind the EGFR binding domains expressed on the surface of the cells. Insome embodiments, the isolated cells are T-cells, an animal T-cell, amammalian T-cell, a feline T-cell, a canine T-cell or a human T-cell,thereby producing EGFR CAR T-cells. In certain embodiments, the isolatedcell is an NK-cell, e.g., an animal NK-cell, a mammalian NK-cell, afeline NK-cell, a canine NK-cell or a human NK-cell, thereby producingEGFR CAR NK-cells.

Sources of T or NK Cells.

Prior to expansion and genetic modification of the T cells of theinvention, a source of T cells is obtained may be obtain from a subjector a culture. T cells can be obtained from a number of sources in asubject, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors.

Methods of isolating relevant cells are well known in the art and can bereadily adapted to the present application; an exemplary method isdescribed in the examples below. Isolation methods for use in relationto this disclosure include, but are not limited to Life TechnologiesDynabeads® system; STEMcell Technologies EasySep™, RoboSep™,RosetteSep™, SepMate™; Miltenyi Biotec MACS™ cell separation kits, andother commercially available cell separation and isolation kits.Particular subpopulations of immune cells may be isolated through theuse of beads or other binding agents available in such kits specific tounique cell surface markers. For example, MACS™ CD4+ and CD8+ MicroBeadsmay be used to isolate CD4+ and CD8+ T-cells.

Alternatively, cells may be obtained through commercially available cellcultures, including but not limited to, for T-cells, lines BCL2 (AAA)Jurkat (ATCC® CRL-2902™), BCL2 (S70A) Jurkat (ATCC® CRL-2900™), BCL2(S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL-2899™), NeoJurkat (ATCC® CRL-2898™); and, for NK cells, lines NK-92 (ATCC®CRL-2407™), NK-92MI (ATCC® CRL-2408™).

Vectors.

CARs may be prepared using vectors. The preparation of exemplary vectorsand the generation of CAR expressing cells using said vectors isdiscussed in detail in the examples below. In summary, the expression ofnatural or synthetic nucleic acids encoding CARs is typically achievedby operably linking a nucleic acid encoding the CAR polypeptide orportions thereof to a promoter, and incorporating the construct into anexpression vector. The vectors can be suitable for replication andintegration eukaryotes.

Methods for producing cells comprising vectors and/or exogenous nucleicacids are well-known in the art. See, for example, Sambrook et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York). Regardless of the method used to introduceexogenous nucleic acids into a host cell or otherwise expose a cell tothe inhibitor of the present invention, in order to confirm the presenceof the recombinant DNA sequence in the host cell, a variety of assaysmay be performed. Such assays include, for example, “molecularbiological” assays well known to those of skill in the art, such asSouthern and Northern blotting, RT-PCR and PCR; “biochemical” assays,such as detecting the presence or absence of a particular peptide, e.g.,by immunological means (ELISAs and Western blots) or by assays describedherein to identify agents falling within the scope of the invention.

In some embodiments, the isolated nucleic acid sequence encodes for aCAR comprising, or alternatively consisting essentially of, or yetfurther consisting of an antigen binding domain of an anti-EGFR antibodythat recognizes either or both wt and mutant EGFR, a CD28 costimulatorysignaling region and/or a 4-1BB costimulatory signaling region, and aCD3 zeta signaling domain. In specific embodiments, the isolated nucleicacid sequence comprises, or alternatively consisting essentiallythereof, or yet further consisting of, sequences encoding an antigenbinding domain of an anti-EGFR antibody followed by a hinge region, aCD28 costimulatory signaling region and/or a 4-1BB costimulatorysignaling region followed by a CD3 zeta signaling domain. In a furtheraspect, the antigen binding domain comprises a HC variable region and aLC variable region that is optionally connected by a linker polypeptide.

In some embodiments, the isolated nucleic acid comprises apolynucleotide conferring antibiotic resistance.

In some embodiments, the isolated nucleic acid sequence is comprised ina vector. In certain embodiments, the vector is a plasmid. In otherembodiments, the vector is a viral vector. In specific embodiments, thevector is a lentiviral vector.

The preparation of exemplary vectors and the generation of CARexpressing cells using said vectors is discussed in detail in theexamples below. In summary, the expression of natural or syntheticnucleic acids encoding CARs is typically achieved by operably linking anucleic acid encoding the CAR polypeptide or portions thereof to apromoter, and incorporating the construct into an expression vector. Thevectors can be suitable for replication and integration eukaryotes.Methods for producing cells comprising vectors and/or exogenous nucleicacids are well-known in the art. See, for example, Sambrook et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York).

In one aspect, the term “vector” intends a recombinant vector thatretains the ability to infect and transduce non-dividing and/orslowly-dividing cells and integrate into the target cell's genome. Inseveral aspects, the vector is derived from or based on a wild-typevirus. In further aspects, the vector is derived from or based on awild-type lentivirus. Examples of such, include without limitation,human immunodeficiency virus (HIV), equine infectious anemia virus(EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiencyvirus (FIV). Alternatively, it is contemplated that other retrovirus canbe used as a basis for a vector backbone such murine leukemia virus(MLV). It will be evident that a viral vector according to thedisclosure need not be confined to the components of a particular virus.The viral vector may comprise components derived from two or moredifferent viruses, and may also comprise synthetic components. Vectorcomponents can be manipulated to obtain desired characteristics, such astarget cell specificity.

The recombinant vectors of this disclosure are derived from primates andnon-primates. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of human acquiredimmunodeficiency syndrome (AIDS), and the simian immunodeficiency virus(SIV). The non-primate lentiviral group includes the prototype “slowvirus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV). Prior art recombinantlentiviral vectors are known in the art, e.g., see U.S. Pat. Nos.6,924,123; 7,056,699; 7,07,993; 7,419,829 and 7,442,551, incorporatedherein by reference.

U.S. Pat. No. 6,924,123 discloses that certain retroviral sequencefacilitate integration into the target cell genome. This patent teachesthat each retroviral genome comprises genes called gag, pol and envwhich code for virion proteins and enzymes. These genes are flanked atboth ends by regions called long terminal repeats (LTRs). The LTRs areresponsible for proviral integration, and transcription. They also serveas enhancer-promoter sequences. In other words, the LTRs can control theexpression of the viral genes. Encapsidation of the retroviral RNAsoccurs by virtue of a psi sequence located at the 5′ end of the viralgenome. The LTRs themselves are identical sequences that can be dividedinto three elements, which are called U3, R and U5. U3 is derived fromthe sequence unique to the 3′ end of the RNA. R is derived from asequence repeated at both ends of the RNA, and U5 is derived from thesequence unique to the 5′end of the RNA. The sizes of the three elementscan vary considerably among different retroviruses. For the viralgenome, the site of poly (A) addition (termination) is at the boundarybetween R and U5 in the right hand side LTR. U3 contains most of thetranscriptional control elements of the provirus, which include thepromoter and multiple enhancer sequences responsive to cellular and insome cases, viral transcriptional activator proteins.

With regard to the structural genes gag, pol and env themselves, gagencodes the internal structural protein of the virus Gag protein isproteolytically processed into the mature proteins MA (matrix), CA(capsid) and NC (nucleocapsid). The pol gene encodes the reversetranscriptase (RT), which contains DNA polymerase, associated RNase Hand integrase (IN), which mediate replication of the genome.

For the production of viral vector particles, the vector RNA genome isexpressed from a DNA construct encoding it, in a host cell. Thecomponents of the particles not encoded by the vector genome areprovided in trans by additional nucleic acid sequences (the “packagingsystem”, which usually includes either or both of the gag/pol and envgenes) expressed in the host cell. The set of sequences required for theproduction of the viral vector particles may be introduced into the hostcell by transient transfection, or they may be integrated into the hostcell genome, or they may be provided in a mixture of ways. Thetechniques involved are known to those skilled in the art.

Retroviral vectors for use in this disclosure include, but are notlimited to Invitrogen's pLenti series versions 4, 6, and 6.2 “ViraPower”system. Manufactured by Lentigen Corp.; pHIV-7-GFP, lab generated andused by the City of Hope Research Institute; “Lenti-X” lentiviralvector, pLVX, manufactured by Clontech; pLKO.1-puro, manufactured bySigma-Aldrich; pLemiR, manufactured by Open Biosystems; and pLV, labgenerated and used by Charité Medical School, Institute of Virology(CBF), Berlin, Germany.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentdisclosure, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the disclosure.

Packaging Vector and Cell Lines.

CARs can be packaged into a retroviral packaging system by using apackaging vector and cell lines. The packaging plasmid includes, but isnot limited to retroviral vector, lentiviral vector, adenoviral vector,and adeno-associated viral vector. The packaging vector containselements and sequences that facilitate the delivery of genetic materialsinto cells. For example, the retroviral constructs are packagingplasmids comprising at least one retroviral helper DNA sequence derivedfrom a replication-incompetent retroviral genome encoding in trans allvirion proteins required to package a replication incompetent retroviralvector, and for producing virion proteins capable of packaging thereplication-incompetent retroviral vector at high titer, without theproduction of replication-competent helper virus. The retroviral DNAsequence lacks the region encoding the native enhancer and/or promoterof the viral 5′ LTR of the virus, and lacks both the psi functionsequence responsible for packaging helper genome and the 3′ LTR, butencodes a foreign polyadenylation site, for example the SV40polyadenylation site, and a foreign enhancer and/or promoter whichdirects efficient transcription in a cell type where virus production isdesired. The retrovirus is a leukemia virus such as a Moloney MurineLeukemia Virus (MMLV), the Human Immunodeficiency Virus (HIV), or theGibbon Ape Leukemia virus (GALV). The foreign enhancer and promoter maybe the human cytomegalovirus (HCMV) immediate early (IE) enhancer andpromoter, the enhancer and promoter (U3 region) of the Moloney MurineSarcoma Virus (MMSV), the U3 region of Rous Sarcoma Virus (RSV), the U3region of Spleen Focus Forming Virus (SFFV), or the HCMV IE enhancerjoined to the native Moloney Murine Leukemia Virus (MMLV) promoter. Theretroviral packaging plasmid may consist of two retroviral helper DNAsequences encoded by plasmid based expression vectors, for example wherea first helper sequence contains a cDNA encoding the gag and polproteins of ecotropic MMLV or GALV and a second helper sequence containsa cDNA encoding the env protein. The Env gene, which determines the hostrange, may be derived from the genes encoding xenotropic, amphotropic,ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virusenv proteins, or the Gibbon Ape Leukemia Virus (GALV env protein, theHuman Immunodeficiency Virus env (gp160) protein, the VesicularStomatitus Virus (VSV) G protein, the Human T cell leukemia (HTLV) typeI and II env gene products, chimeric envelope gene derived fromcombinations of one or more of the aforementioned env genes or chimericenvelope genes encoding the cytoplasmic and transmembrane of theaforementioned env gene products and a monoclonal antibody directedagainst a specific surface molecule on a desired target cell.

In the packaging process, the packaging plasmids and retroviral vectorsexpressing the EGFR are transiently cotransfected into a firstpopulation of mammalian cells that are capable of producing virus, suchas human embryonic kidney cells, for example 293 cells (ATCC No.CRL1573, ATCC, Rockville, Md.) to produce high titer recombinantretrovirus-containing supernatants. In another method of the inventionthis transiently transfected first population of cells is thencocultivated with mammalian target cells, for example human lymphocytes,to transduce the target cells with the foreign gene at highefficiencies. In yet another method of the invention the supernatantsfrom the above described transiently transfected first population ofcells are incubated with mammalian target cells, for example humanlymphocytes or hematopoietic stem cells, to transduce the target cellswith the foreign gene at high efficiencies.

In another aspect, the packaging plasmids are stably expressed in afirst population of mammalian cells that are capable of producing virus,such as human embryonic kidney cells, for example 293 cells. Retroviralor lentiviral vectors are introduced into cells by either cotransfectionwith a selectable marker or infection with pseudotyped virus. In bothcases, the vectors integrate. Alternatively, vectors can be introducedin an episomally maintained plasmid. High titer recombinantretrovirus-containing supernatants are produced.

Activation and Expansion of Tor NK Cells.

Whether prior to or after genetic modification of the T cells or NKcells to express a desirable CAR, the T cells or NK cells can beactivated and expanded generally using generally known methods such asthose described in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680;6,692,964; 5,858,358; 6,887,466; 6,905,681; 7, 144,575; 7,067,318; 7,172,869; 7,232,566; 7, 175,843; 5,883,223; 6,905,874; 6,797,514;6,867,041. Methods of activating relevant cells are well known in theart and can be readily adapted to the present application; an exemplarymethod is described in the examples below. Stimulation with the EGFRantigen ex vivo can activate and expand the selected CAR expressingT-cell subpopulation.

Alternatively, the T-cells may be activated in vivo by interaction withEGFR antigen. Thus, in a further aspect, this disclosure provides anisolated expanded population of cells expressing an EGFR CAR.

Isolation methods for use in relation to this disclosure include, butare not limited to Life Technologies Dynabeads® system activation andexpansion kits; BD Biosciences Phosflow™ activation kits, MiltenyiBiotec MACS™ activation/expansion kits, and other commercially availablecell kits specific to activation moieties of the relevant cell.Particular subpopulations of immune cells may be activated or expandedthrough the use of beads or other agents available in such kits. Forexample, α-CD3/α-CD28 Dynabeads® may be used to activate and expand apopulation of isolated T-cells.

As disclosed above, chimeric antigen receptors comprise an antigenrecognition moiety and a cell activation moiety. Aspects of the presentdisclosure related to a chimeric antigen receptor (CAR) comprising anantigen binding domain specific to wildtype and mutant EGFR.

Methods of Use

Therapeutic Application.

The CAR T-cells and NK cells of the present disclosure can be used totreat tumors and cancers that express EGFR, such as glioblastoma. TheEGFR CAR cells of the present disclosure can be administered eitheralone or in combination with diluents, known anti-cancer therapeutics(chemotherapeutics, surgery and/or radiation), and/or with othercomponents such as cytokines or other cell populations that areimmunostimulatory. The methods of this disclosure can be first-line,second-line, third-line or fourth line therapy.

Method aspects of the present disclosure relate to methods forinhibiting the growth of a tumor in a subject in need thereof and/or fortreating a cancer patient in need thereof. In some embodiments, thetumor is a solid tumor. In some embodiments, the tumors/cancer isglioblastoma or a glioblastoma stem cell. In certain embodiments, thesemethods comprise, or alternatively consist essentially of, or yetfurther consist of, administering to the subject or patient an effectiveamount of the isolated cell as described herein, e.g., the isolated cellthat comprises an EGFR CAR. In still further embodiments, the isolatedcell is a T-cell or an NK cell. In some embodiments, the isolated cellis autologous to the subject or patient being treated. In a furtheraspect, the tumor expresses EGFR antigen and the subject has beenselected for the therapy by a diagnostic, such as the one describedherein. In a further aspect, the isolated cells are directly injected orinfused into the brain of the subject to inhibit the growth of aglioblastoma or glioblastoma stem cells.

Pharmaceutical compositions comprising the EGFR CAR of the presentinvention may be administered in a manner appropriate to the disease tobe treated or prevented. Any appropriate method of administration can beused, e.g., direct injection and/or systemically such as by intravenousinjection. The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials. The method of inhibiting the growth ofa tumor can be applied to a subject including but not limited to human,dog, cat, horse, and other species.

In one embodiment, the disclosure provides a method for determining if apatient is likely to respond or is not likely to EGFR CAR therapy, themethod comprising, or alternatively consisting essentially of, or yetfurther consisting of contacting a tumor sample isolated from thepatient with an effective amount of an EGFR antibody and detecting thepresence of any antibody bound to the tumor sample, wherein the presenceof antibody bound to the tumor sample indicates that the patient islikely to respond to the EGFR CAR therapy and the absence of antibodybound to the tumor sample indicates that the patient is not likely torespond to the EGFR CAR therapy. In another embodiment, the methodfurther comprises administering an effective amount of the EGFR CARtherapy to the patient that is determined likely to respond to the EGFRCAR therapy. In this method, the patient can suffer from a brain cancer.In some embodiments, this brain cancer is glioblastoma or a metastasisof another cancer (e.g. breast cancer). In one aspect, the sample isobtained from a subject that is diagnosed as having, suspected ashaving, or at risk of having cancer.

In one aspect, the detection comprises, or alternatively consistsessentially of, or yet further consists of one or more ofimmunohistochemistry (IHC), Western blotting, Flow cytometry or ELISA.

In one aspect, the method comprises, or alternatively consistsessentially of, or yet further consists of isolating the biologicalsample from the subject. In a further aspect, T cells or NK cells areisolated from the subject, transduced with an isolated nucleic acidencoding the EGFR CAR and an expanded population of transduced cells areprepare for administration to the subject using a method as describedherein.

In one aspect, the subject is a mammal, such as a human patient.

Compositions and Carriers

Additional aspects of the invention relate to compositions comprising acarrier and one or more of the products—e.g., EGFR CAR, an isolated cellcomprising a EGFR CAR, an isolated nucleic acid encoding the EGFR CAR ortheir complements and/or a vector comprising the isolated nucleic acid.The carriers can be solid carriers or liquid carriers such aspharmaceutically acceptable carriers.

Briefly, pharmaceutical compositions of the present invention includingbut not limited to any one of the claimed compositions that in oneaspect comprises a cell population as described herein, in combinationwith one or more pharmaceutically or physiologically acceptablecarriers, diluents or excipients or solid supports. Such compositionsmay comprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present disclosure may be formulated for oral, intravenous, topical,enteral, and/or parenteral administration. In certain embodiments, thecompositions of the present disclosure are formulated for intravenousadministration.

Briefly, pharmaceutical compositions of the present invention includingbut not limited to any one of the claimed compositions as describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated or prevented. Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

Combination Therapies

Aspects of this disclosure relate to combination therapies involving theEGFR CAR expressing cells disclosed herein and an oncolytic herpessimplex virus. Clinical studies have demonstrated that oncolytic herpesviruses, when administered alone, work with limited efficacy. Theexperiments and disclosure provided herein demonstrate promising resultsfor EGFR CAR expressing cells. Applicants have found that theadministration of both EGFR CAR expressing cells and an oncolytic herpessimplex has a synergistic effect. Not to be bound by theory, it issuspected that EGFR CAR expressing cells may facilitate the destructionof the tumor tissue structure making cancer cells more accessible forinfection and destruction by the oncolytic herpes viruses.

In some embodiments, the EGFR CAR expressing cells contemplated for usein a combination therapy are isolated immune cells, optionally selectedfrom T cells or NK cells. In some embodiments, these EGFR CAR expressingcells are irradiated. In some embodiments, the EGFR CAR expressing cellsare irradiated at a dose between 1 and 10000 cGy, such as (but notlimited to) greater than, less thank, or about 1 cGy, 5 cGy, 10 cGy, 50cGy, 100 cGy, 500 cGy, 1000 cGy, 5000 cGy, or 10000 cGy. In someembodiments, the irradiation dose is optimize the proliferation of theEGFR CAR expressing cells while maintaining their full activity up togreater than or about 12, 24, 36, 48, 60, 72, 84, 96, or more hours. Insome embodiments, the EGFR CAR expressing cells are modified (throughirradiation or other means) in order not to eradicate or eliminate oHSV.

In some embodiments the oncolytic herpes simplex virus is one or more ofthe oncolytic herpes viruses that have undergone clinical trials and aredisclosed herein above. In some embodiments, the oncolytic herpessimplex virus is derived from HSV-1 or HSV-2.

In some embodiments, the EGFR CAR expressing cells are administered atthe same time, before, or after the oncolytic herpes simplex virus. Insome embodiments, the EGFR CAR expressing cells are administered first.In other embodiments, the oncolytic herpes simplex virus is administeredfirst. In some embodiments, the EGFR CAR expressing cells areadministered in single or multiple doses. In some embodiments theoncolytic herpes simplex viruses is administered in single or multipledoses. In one embodiment, the EGFR CAR expressing cells are administeredfirst followed by administration of the oncolytic herpes simplex virusesover one or more days.

Kits

Further provided herein are kits comprising one or more of the EGFR CARas described herein, an isolated nucleic acid encoding the EGFR CAR ortheir complements, a vector comprising the nucleic acids, an isolatedcell as described herein, or an isolated complex as described herein.

The following examples are intended to illustrate and not limit thisdisclosure.

Experiment 1—Generation and Characterization of EGFR CAR ExpressingCells Cell Culture

Human GB cell lines [Gli36DeltaEGFR (Gli36dEGFR), U251, and LN229] andhuman patient-derived GB stem cells (GB 1123, GB30, GB83, and GB326)(Mao, P. et al. (2013) Proc Natl Acad Sci. USA 110:8644-8649) were usedin this study. GB84V3SL, GB157V3SL, and GB19 were also established fromGB patients using the same protocol (Mao, P. et al. (2013) Proc NatlAcad Sci. USA 110:8644-8649). All GB cell lines, 293T cells and Phoenixcells were cultured in DMEM (Invitrogen, Grand Island, N.Y.)supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100μg/ml). GB stem cells were cultured in DMEM-F-12 medium supplementedwith bFGF (20 ng/ml), EGF (20 ng/ml), Glutamax (1:100), B27 (1:100),heparin (5 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml).All stocks of the above antibiotics and cytokine stocks were purchasedfrom Invitrogen. Human NK cell lines NK-92 and NKL were maintained inRPMI-1640 (Invitrogen) supplemented with 20% FBS, penicillin (100 U/ml),streptomycin (100 μg/ml) and 150 IU/mL recombinant human (rh) 1L-2(Hoffman-La Roche Inc., Nutley, N.J.).

Mice

Six to eight-week-old NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ mice (NSG)mice were obtained from Jackson Laboratories (Bar Harbor, Me.). Allanimal work was approved by The Ohio State University Animal Care andUse Committee and the methods were carried out in accordance with theapproved guidelines. Mice were monitored frequently for GB diseaseprogression, and sacrificed when they became moribund with neurologicimpairments and obvious weight loss.

Generation of Anti-EGFR CAR Lentiviral Construct

The anti-EGFR single chain variable fragment (scFv) was derived from ahybridoma cell line that produces mouse monoclonal antibody 528recognizing both wtEGFR and EGFRvIII (Hayashi, H. et al. (2004) CancerImmunology, Immunotherapy: CII 53:497-509; Humphrey, P. A. et al. (1990)Proc Natl Acad Sci. USA 87:4207-4211). The coding domain sequences forvariable regions of heavy (VH) and light (VL) chains were amplifiedseparately and assembled using a linker by overlapping PCR reaction. TheVH-linker-VL fragment was incorporated in frame with CD28-CD3ζ portion(Pule, M. A. et al. (2005) Mol Ther. 12:933-941) that was incised from aretroviral vector. The entire anti-EGFR-scFv-CD28-CD3ζ fragment was thenligated into a lentiviral vector designated pCDH-CMV-MCS-EF1-copGFP(pCDH, System Biosciences, Mountain View, Calif.) to generate apCDH-EGFR-scFv-CD28-CD3ζ (pCDH-EGFR-CAR) construct.

Lentivirus Production and Transduction of NK Cells

To produce lentiviruses for infection of NK cells (NK-92 and NKL), 293Tcells were co-transfected with the aforementionedpCDH-EGFR-scFv-CD28-CD3 plasmid or a mock pCDH vector together with thepackaging constructs pCMV-VSVG and pCMV-DR9 using calcium phosphatetransfection reagent (Promega, Madison, Wis.). The transfection andinfection procedures were modified from a previously published protocol(Chu, J. et al. (2014) Leukemia 28:917-927).

Generation of G119 Stem Cells Stably Expressing wtEGFR or EGFRvIII

Phoenix cells were co-transfected with the pBABE-wtEGFR (a wtEGFRexpression construct), or pBABE-EGFRvIII (an EGFRvIII expressionconstruct) or a pBABE empty vector together with Sara3 packaging plasmidusing calcium phosphate transfection reagent (Promega, Madison, Wis.).Two days after transfection, the infectious supernatants were harvestedto infect GB19 stem cells in the presence of polybrene (8 μg/mL).Retrovirus was produced in serum-free DMEM-F12 medium for infection ofGB 19 stem cells, and GFP positive GB19-wtEGFR or GB19-EGFRvIII cellswere then sorted using a FACS Aria II cell sorter (BD Biosciences, SanJose, Calif.).

Flow Cytometry

To evaluate NK cell surface expression of EGFR-CAR, transduced NK cellswere washed with PBS containing 4% BSA, and incubated withbiotin-labeled goat anti-mouse F(ab′)₂ polyclonal antibody or normalpolyclonal goat IgG antibody (Jackson ImmunoResearch, West Grove, Pa.)as an isotype control as described previously (Chu, J. et al. (2014)Leukemia 28:917-927). Then cells were washed again and stained withallophycocyanin (APC)-conjugated streptavidin (Jackson ImmunoResearch,West Grove, Pa.). To determine wtEGFR and/or EGFRvIII expression on thesurface of GB cells, the cells were incubated with mouse monoclonalanti-human EGFR (clone H11, DAKO, Carpinteria, Calif.) which recognizesboth wild-type EGFR and its mutant form (EGFRvIII), followed by stainingwith APC conjugated goat anti-mouse IgG second antibody.

Reverse Transcription PCR

To detect EGFR mRNA expression in glioma cells, RNA was extracted fromthe cell lines with RNeasy Mini Kit (Qiagen, Hilden, Germany) andquantified with NanoDrop (Thermo Fisher, Wilmington, Del.). Reversetranscript was produced using M-MLV reverse transcriptase (Invitrogen,Grand Island, N.Y.), and PCR was conducted with GoTaq® Flexi DNAPolymerase (Promega, Madison, Wis.). The forward primer isTGACTCCGTCCAGTATTGATCG, and the reverse primer isGCCCTTCGCACTTCTTACACTT. The PCR reaction parameters are 95° C. 5 min, 35cycles at 95° C. 40s, 55° C. 40s, 72° C. 1 min, and final extension at72° C. for 10 min.

Cytotoxicity Assay

A standard 4-h ⁵¹Cr release assay was performed as described previously(Yu, J. et al. (2010) Blood 115:274-281). Briefly, target cells werelabeled with ⁵¹Cr and co-cultured with modified NK cells at variouseffector/target ratios (E/T) in the wells of 96-well V-bottom plates at37° C. for 4 h. Supernatants were harvested and transferred intoscintillation vials containing a liquid scintillation cocktail (FisherScientific, Waltham, Mass.), and the release of ⁵¹Cr was measured onBeckman Liquid Scintillation Counter LS-6500. Target cells incubated incomplete medium or 1% SDS were used to determine spontaneous or maximal⁵¹Cr release, respectively. Percentage of specific cell lysis wascalculated using the standard formula: 100×(cpm experimental release−cpmspontaneous release)/(cpm maximal release−cpm spontaneous release).

IFN-γ Release Assay

1×10⁶ target cells were incubated with equal numbers of NK effectorcells in 96-well V bottom plates for 24 h. Cell-free supernatants wereassayed for IFN-γ secretion by enzyme-linked immunosorbent assay (ELISA)using a kit from R&D Systems (Minneapolis, Minn.) in accordance with themanufacturer's protocol. Data depicted in figures represent mean valuesof triplicate wells from one of three representative experiments withsimilar results.

Treatment of Orthotopic Human GB30 Xenografts in NSG Mice

GB30 GB stem cells were retrovirally transduced with Pinco-pGL3-luc/GFPvirus expressing firefly luciferase (FFL) as previously described (He,S. et al. (2013) Blood 121:4663-4671). GFP positive cells were sortedusing a FACS Aria II cell sorter (BD Biosciences, San Jose, Calif.), andwere designated “GB30-FFL” cells. NSG mice were anesthetized and fixedin a stereotactic apparatus, a burr hole was drilled 2 mm lateral and 1mm anterior to the bregma to a depth of 3.25 mm, and 5×10⁴ GB30-FFLcells in 2 μl Hank's buffered salt solution (HBSS) were implanted. OnDay 7, the mice were intracranially injected with 2×10⁶ effector cells(non-irradiated), i.e. EGFR-CAR-transduced NK-92 cells (NK-92-EGFR-CAR)or mock-transduced NK-92 cells (NK-92-EV) in 5 td HBSS. Mice treatedwith 5 μl HBSS only were used as control. Mice were monitored daily andeuthanized when showing signs of morbidity. Two weeks after GB30-FFLcell inoculation, the mice were intraperitoneally (i.p.) infused withD-luciferin (150 mg/kg body weight; Gold Biotechnology, St. Louis, Mo.),anesthetized with isoflurane, and imaged using In Vivo Imaging System(IVIS-100, PerkinElmer, Waltham, Mass.) with living image software(PerkinElmer).

Treatment of Orthotopic Human U251 Xenografts in NSG Mice

U251 GB cells were retrovirally transduced with Pinco-pGL3-luc/GFP virusexpressing firefly luciferase (FFL) as previously described (He, S. etal. (2013) Blood 121:4663-4671). GFP positive cells were sorted using aFACS Aria II cell sorter (BD Biosciences, San Jose, Calif.), and weredesignated “U251-FFL” cells. NSG mice were anesthetized and fixed in astereotactic apparatus, and a burr hole was drilled 2 mm lateral and 1mm anterior to the bregma to a depth of 3.25 mm, through which 10⁵U251-FFL cells in 2 μl Hank's buffered salt solution (HBSS) wereinoculated on day 0. On day 10, 40, 70 the mice were intracraniallyinjected with 2×10⁶ effector cells, i.e. EGFR-CAR-transduced NK-92 cells(NK-92-EGFR-CAR) or mock-transduced NK-92 cells (NK-92-EV) in 5 μl HBSS.Mice treated with 5 μl HBSS only were used as control. Mice weremonitored daily and euthanized when showing signs of morbidity. On day100 after U251-FFL cell inoculation, the mice were intraperitoneally(i.p.) infused with D-luciferin (150 mg/kg body weight; GoldBiotechnology, St. Louis, Mo.), anesthetized with isoflurane, and imagedusing In Vivo Imaging System (IVIS-100, PerkinElmer, Waltham Mass., USA)with living image software (PerkinElmer).

Organ and Tissue Distribution Assays of NK-92-EGFR-CAR Cells

2×10⁶ NK-92 EGFR-CAR cells were intracranially injected into threeGB30-bearing mice 7 days after tumor implantation. Three days later, allmice were sacrificed and liver, lung, spleen, bone marrow, blood, andbrain were harvested. Half of organs or tissues were used for genomicDNA isolation with a DNA isolation kit (Invitrogen, Carlsbad, Calif.),and PCR was performed with primers to amplify the EGFR-CAR scFv region.The PCR forward primer was AGGTCACTCTGACTGTAGACA, and the reverse primerwas GTTCATGTAGTCACTGTGCAG. The PCR reaction parameters were 95° C. 5min, 35 cycles at 95° C. 40 s, 55° C. 40 s, 72° C. 1 min, and finalextension at 72° C. for 10 min.

The other half of organs or tissues was used to isolate mononuclearimmune cells using density gradient centrifugation in Percoll (GEHealthcare, Pittsburgh, Pa.). The collected immune cells were surfacestained with V450 mouse anti-human CD3e and APC mouse anti-human CD56antibodies (eBioscience, San Diego, Calif.) to determine the presence ofNK-92-EGFR-CAR cells in a specific organ or tissue.

Statistical Analysis

Unpaired Student's t test was utilized to compare two independent groupsfor continuous endpoints if normally distributed. One-way ANOVA was usedwhen three or more independent groups were compared. For non-normallydistributed endpoints, such as in vivo bioluminescence intensity, aKruskal-Wallis test was utilized to compare the median of NK-92-EGFR-CARgroup to NK-92-EV and HBSS-treated groups. For survival data,Kaplan-Meier curves were plotted and compared using a log-rank test. Alltests are two-sided. P¹ values were adjusted for multiple comparisonsusing Bonferroni method. P values less than 0.05 were consideredsignificant

Generation of 293T Cell Lines Stably Expressing wtEGFR or EGFRvIII.

Phoenix cells were co-transfected with the pBABE-wtEGFR (wt EGFRexpression construct), pBABE-EGFRvIII (EGFRvIII expression construct),or pBABE empty vector together with Sara3 packaging plasmid usingcalcium phosphate transfection reagent (Promega, Madison, Wis., USA).Two days after transfection, supernatants were harvested to infect 293Tcells in the presence of polybrene (8 μg/mL). GFP positive cells weresorted using a FACS Aria II cell sorter (BD Biosciences, San Jose,Calif., USA).

Lentivirus Production and Transduction of Primary NK Cells.

Lentiviruses were produced as previously described (Chu, J. et al.(2014) Leukemia 28:917-927) and were concentrated by ultracentrifuge at20,000 g for 90 minutes at 4° C. and resuspended with 1× PBS. Humanprimary NK cells were isolated from peripheral blood leukopacks ofhealthy donors (American Red Cross, Columbus, Ohio) as describedpreviously (He, S. et al. (2013) Blood 121:4663-4671) and infected withlentiviruses (MOI=2.5) by three consecutive rounds of centrifugation at2000 rpm and 32° C. for 2 h (gentle resuspension between rounds), andGFP positive cells were sorted using a FACS Aria II cell sorter (BDBiosciences, San Jose, Calif., USA). Standard 4-h ⁵¹Cr release assayswere performed to evaluate the cytotoxicity of primary NK cellstransduced with EGFR-CAR plasmid or control vector against U251 andGli36dEGFR GB cell lines and GB30 and GB157V3SL patient-derived GB stemcells.

Antibody Blocking Assay.

GB30 stem cells and U251 cell line were pretreated with 10 μg/ml EGFRneutralizing antibody (clone 528, EMD Millipore, Billerica, Mass.) or anisotype-matched antibody at 37° C. for 30 minutes. Standard 4-h 51Crrelease assays were then performed to evaluate cytotoxicity ofNK-92-EGFR-CAR cells and control cells against the 528 antibody- orIgG-pretreated target cells. IFN-γ secretion was also quantified byELISA on cell-free supernatants after 1×10⁶ target cells pretreated with528 antibody- or IgG were incubated with an equal number ofNK-92-EGFR-CAR cells or control cells in 96-well V bottom plates for 24h.

Hematoxylin and Eosin (H&E) Staining of Brain Sections of GB30-BearingMice.

NSG mice were intracranially injected with 5×10⁴ GB30 cells on day 0. Onday 7, the m ice were intracranially injected with 2×10⁶ NK-92-EGFR-CARcells in 5 W HBSS. On day 10, the mice were sacrificed and brain tissueswere harvested and immersed in 10% formalin for 24 h, and then brainswere embedded in paraffin and processed for H&E staining.

Results Expression of EGFR or EGFRvIII on GB Cell Lines andPatient-Derived GB Stem Cells

To assess the surface expression of wtEGFR or EGFRvIII on a panel of GBcell lines and patient-derived GB stem cells, intact cells were stainedwith an EGFR-specific antibody that recognizes both wtEGFR and EGFRvIII,followed by flow cytometric analysis. As shown in FIG. 1A, EGFR wasexpressed on the surface of GB cell lines (Gli36dEGFR, U251, and LN229),patient-derived GB mesenchymal (MES) stem cells (GB30, GB83, GB1123 andGB326) and GB proneural (PN) stem cells (GB84V3S and GB157V3SL). Tofurther address whether wtEGFR or EGFRvIII transcript was expressed inthese cells, Applicants carried out RT-PCR with specific primers andobserved that wtEGFR mRNA was expressed in two GB cell lines, U251 andLN229. EGFRvIII mRNA was detectable in the GB cell line Gli36dEGFR, andin six GB stem cells that Applicants generated from GB patients (Mao, P.et al. (2013) Proc Natl Acad Sci. USA 110:8644-8649): GB30, GB83,GB1123, GB326, GB84V3SL and GB157V3SL (FIG. 1B). In contrast, one GBstem cell, GB19 (proneural), and the two NK cell lines, NK-92 and NKL,had no detectable EGFR expression by flow cytometric analysis or RT-PCR(FIGS. 1A, 1B).

Generation of NK-92 and NKL NK Cells Expressing EGFR-CAR

A second-generation EGFR-specific CAR construct was generated in a pCDHlentiviral vector backbone. This construct sequentially contains asignal peptide (SP), a heavy chain variable region (VH), a linker, alight chain variable region (VL), a hinge, CD28 transmembrane andintracellular domain, and CD3ζ signaling moiety (FIG. 2A). NK-92 and NKLcell lines were transduced with the EGFR-CAR construct to generate NK-92EGFR-CAR and NKL EGFR-CAR cells respectively. The transduced cells weresorted for the expression of GFP expressed by the vector. To validatecell surface expression of EGFR-CAR on the transduced NK-92 and NKLcells, flow cytometric analysis was performed using a goat anti-mouseF(ab′)_(Z) antibody that recognized the scFv portion of anti-EGFR. Thedata from FIG. 2B showed an obvious increase in cell surface EGFR-CARexpression in EGFR-CAR-transduced NK-92 and NKL cells over thosetransduced with empty vector, the latter of which had undetectableEGFR-CAR expression.

Efficacy of EGFR-CAR-Modified NK Cell Cytotoxicity and IFN-γ ProductionAgainst EGFR⁺ GB Cell Lines

The cytotoxicity of EGFR-CAR- and mock-transduced NK cells was assessedagainst GB cells using a standard chromium release assay at varyingratios of effector cells to target cells. FIGS. 3A-3B show significantlyenhanced cytotoxicity of NK-92 EGFR-CAR cells against EGFR⁺ Gli36dEGFR,U251, and LN229 cells compared to control NK-92-EV cells (FIG. 3A, upperpanels). Similar data were observed in experiments using the NKL cellline transduced with EGFR-CAR (FIG. 3A, lower panels). Human primary NKcells transduced with EGFR-CAR also showed significantly more potentcytotoxicity than control cells against EGFR⁺ Gli36dEGFR and U251 GBcell lines (FIG. 8A). To determine if the observed enhanced cytolyticactivity was accompanied by a similar significant increase in IFN-γsecretion, Applicants co-cultured EGFR-CAR NK-92 cells with EGFR⁺ gliomacells (Gli36dEGFR, U251, and LN229) cells for 24 h and measured IFN-γproduction by ELISA. As shown in FIG. 3B, both EGFR-CAR-modified andmock-transduced NK-92 or NKL cells spontaneously produced low ornegligible levels of IFN-γ when incubated alone. Culturing these cellswith EGFR⁺ glioma cells (Gli36dEGFR, U251 and LN229) induced IFN-γ inboth EGFR-CAR and mock-transduced NK-92 or NKL cell lines, withsignificantly higher levels of IFN-γ produced by EGFR-CAR-modified NK-92or NKL cells than by mock-transduced NK-92 or NKL cells, respectively(FIG. 3B). These results are in agreement with the aforementionedcytotoxicity data, and together indicate that modification with EGFR-CARcan significantly enhance NK cell effector functions in response toEGFR⁺ glioma cells.

Efficacy of EGFR-CAR-Modified NK Cell Cytotoxicity and IFN-γ ProductionAgainst EGFR⁺ GB Stem Cells Derived from GB Patients

Applicants next assessed the capacity of EGFR-CAR-modified NK-92 and NKLcells to lyse patient-derived GB stem cells with surface expression ofendogenous EGFR protein. EGFR-CAR-transduced NK-92 cells demonstrated asignificantly enhanced ability to kill EGFR⁺ GB mesenchymal stem cells(GB 1123 and GB30) and GB proneural stem cells (GB157V3SL and GB84V3SL)when compared to mock-transduced NK cells (FIG. 4A, upper panels).Similar data were observed in experiments repeated using NKL cellstransduced with EGFR-CAR (FIG. 4A, lower panels). Primary NK cellstransduced with EGFR-CAR also showed significantly more potentcytotoxicity than control cells against patient-derived stem cells GB30and GB157V3SL, which express EGFRvIII (FIG. 8B). Likewise,EGFR-CAR-transduced NK-92 and NKL cells produced significantly moreIFN-γ when co-cultured with EGFR⁺ GB stem cells and compared tomock-transduced NK-92 (FIG. 4B, upper panels) and NKL cells (FIG. 4B,lower panels). These results indicate that modification of NK cells withan EGFR-CAR can significantly enhance NK cell cytotoxicity and IFN-γproduction against EGFR⁺ GB stem cells compared to unmodified NK cellcontrols.

Enhanced Cytotoxicity and IFN-γ Production of NK-92-EGFR-CAR CellsDepend on EGFR Surface Expression on Target Cells

Applicants next explored whether the enhanced cytolytic activity andTFN-γ production in EGFR-CAR-transduced NK-92 or NKL cells triggered byGB cells relies on cell surface EGFR antigen expression. Of the GB cellstested, only GB19 stem cell did not express either EGFR or EGFRvIII ontheir surface. Thus, Applicants utilized this cell line to investigatewhether forced wtEGFR or EGFRvIII overexpression in GB19 cells wassufficient to alter their sensitivity to EGFR-CAR-transduced NK-92 orNKL cells. For this purpose, Applicants overexpressed either wtEGFR orEGFRvIII on GB19 cells by retroviral infection, confirmed by flowcytometric analysis (FIG. 5A). There was a significant increase in thecytotoxic activity of EGFR-CAR-modified NK-92 and -NKL cells towardsGB19 cells exogenously overexpressing EGFR or EGFRvIII when compared totarget GB19 cells lacking EGFR expression or mock-transduced NK-92 andNKL effector cells (FIG. 5B). Likewise, EGFR-CAR-transduced NK-92 andNKL cells secreted significantly higher levels of IFN-γ when co-culturedwith EGFR- or EGFRvIII-overexpressing GB19 cells when compared to targetGB19 cells lacking overexpression of EGFR or mock-transduced NK-92 andNKL effector cells (FIG. 5C). These results suggest that the increasedrecognition and elimination of wtEGFR- or EGFRvIII-expressing GB cellsby NK-92-EGFR-CAR cells occur in an EGFR-dependent manner. Also, theseresults were consistent with confirmative experiments showing thatforced EGFR expression in 293T cells resulted in increased cytotoxicityand IFN-γ production of NK-92-EGFR-CAR cells compared to NK-92 cellstransduced with the empty vector (FIGS. 9A-9C). Moreover, Applicantspre-treated GB30 and U251 cells with EGFR neutralizing antibody (thesame clone as the scFv origin), followed by co-culture of thepre-treated tumor cells with mock cells or NK-92-EGFR-CAR cells.Chromium-51 release assay showed that the EGFR blocking antibody bluntedthe enhancements of both cytotoxicity and IFN-7 production inNK-92-EGFR-CAR when compared to an isotype-matched control antibody(FIG. 10), further confirming that the effects that Applicantsidentified for NK-92-EGFR-CAR cells are EGFR-dependent.

NK-92-EGFR-CAR Cells Inhibit GB Tumor Growth and Prolong Survival ofTumor-Bearing Mice in Two Orthotopic Xenograft GB Models

To further address the potential therapeutic application ofNK-92-EGFR-CAR cells, Applicants examined their antitumor activity invivo. Applicants established orthotopic glioma by intracraniallyimplanting EGFRvIII-expressing GB30 glioma stem cells which had beengenetically manipulated to express firefly luciferase (GB30-FFL) intothe brains of NSG mice. The expression of firefly luciferase enabled usto monitor the tumor growth via in vivo bioluminescence imaging. Tominimize potential systemic toxicity, Applicants injected theNK-92-EGFR-CAR intratumorally 7 days post tumor cell implantation. Asshown in FIGS. 6A-6B, mice that received either EGFR-CAR- ormock-transduced NK-92 cells had significantly reduced tumor growth asdetermined by bioluminescence imaging, compared to those injected withHBSS. Importantly, however, the reduction in tumor growth wassignificantly greater in mice treated with NK-92-EGFR-CAR cells thanthose treated with mock-transduced NK-92 cells. In agreement with thesedata, mice treated with NK-92-EGFR-CAR cells survived significantlylonger than mice treated with mock-transduced NK-92 cells or HBSS(median survival of 38 vs 23 days between NK-92-EGFR-CAR- andNK-92-EV-treated mice, p<0.01; median survival of 38 vs 17 days betweenNK-92-EGFR-CAR- and HBSS-treated mice, p<0.01) (FIG. 6C). To furtheraddress the therapeutic efficacy of NK-92-EGFR-CAR cells againstwtEGFR-expressing GB tumor, Applicants established an orthotopic GBmodel by intracranially implanting wtEGFR-expressing U251-FFL cells intoNSG mice. Applicants injected NK-92-EGFR-CAR cells, NK-92-EV cells orHBSS as a vehicle control intratumorally 10 days, 40 days and 70 daysafter tumor cell implantation. As shown in FIGS. 7A-7B, mice which wereinjected intratumorally with EGFR-CAR cells had significantly decreasedtumor burden, compared to those infused with HBSS or NK-92-EV cells.Moreover, mice treated with NK-92-EGFR-CAR cells survived significantlylonger than those receiving NK-92-EV cells or HBSS (median survival of187 vs 150 days between NK-92-EGFR-CAR- and NK-92-EV cell-treated mice,p<0.05; median survival of 187 vs 138 days between NK-92-EGFR-CAR- andHBSS-treated mice, p<0.01) (FIG. 7C). Taken together, NK-92-EGFR-CARcells could efficiently target and eliminate either wtEGFR- orEGFRvIII-expressing GB in vivo.

Assessment of NK-92-EGFR-CAR Cell Migration after Intracranial Injection

To evaluate the safety of intracranial injection of NK-92-EGFR-CARcells, Applicants first analyzed the systemic cell distribution afterintracranial injection. Applicants undertook a flow cytometric analysisand a more sensitive PCR approach to assess the distribution ofNK-92-EGFR-CAR cells in a variety of organs and tissues harvested threedays after their intracranial injection into GB30-bearing mice. As shownin FIG. 6D, CD56+ cells could be identified only in the brain andconstituted only 10.2% of total immune infiltrating cells in the brain.Similarly, PCR analysis was unable to detect EGFR-CAR expression in anyorgan site tested other than brain (FIG. 6E). Together, these datasuggest that intracranial administration of NK-92-EGFR-CAR cells in anorthotopic mouse model of human GB can be performed without extracranialmigration of the effector cells. Applicants next performed Hematoxylinand Eosin (H&F) staining of brain sections of GB30-bearing mice beingintratumorally infused with NK-92-EGFR-CAR cells. The results showedthat NK-92-EGFR-CAR cells distributed only inside the tumor (FIG. 11).

Discussion

In this study, Applicants developed a novel and promising strategyagainst GB, utilizing EGFR-CAR-modified human NK-92 cells tointracranially target human GB. Tumors from GB patients express eitherwtEGFR or both wtEGFR and EGFRvIII (Fan, Q. W. et al. (2013) Cancer Cell24:438-449), suggesting that targeting both forms of this surfaceprotein will have a broader application or be more effective thantargeting only one. Applicants also demonstrated the in vivo efficacyand safety of intracranial injection of EGFR-CAR-modified NK-92 cells inApplicants' orthotopic preclinical model.

CAR T cells have been effectively used for treatment of refractorychronic lymphocytic leukemia and acute lymphoblastic leukemia, andrepresent a powerful new therapeutic modality for these highlydrug-resistant tumors (Porter, D. L. et al. (2011) N Engl J Med.365:725-733; Grupp, S. A. et al. (2013) N Engl J Med. 368:1509-1518;Brentjens, R. J. et al. (2013) Sci Transl Med. 5:177ra138; Papapetrou,E. P. et al. (2011) Nature Biotechnology 29:73-78). Also, severalstudies have demonstrated the use of CAR T cells to treat GB (Morgan, R.A. et al. (2012) Hum Gene Ther. 23:1043-1053; Ohno, M. et al. (2010)Cancer Sci. 101:2518-2524; Ahmed, N. et al. (2010) Clin Cancer Res.16:474-485). But these studies only focused on targeting EGFRvIII.Moreover, CAR T cells can cause cytokine-related adverse events andtumor lysis syndrome (Grupp, S A. et al (2013) N Engl J Med.368:1509-1518; Morgan, R A et al. (2010) Mol Ther. 18:843-851), whichmay result in substantial toxicity or death of patients. In addition,production of autologous CAR T cells is an expensive and time-consumingapproach. Thus, utility of CAR NK cells to target both wtEGFR andEGFRvIII for GB treatment is a good alternative approach.

CAR-engineered NK cell lines (e.g. NK-92 used in this study) couldpotentially provide an off-the-shelf, renewable product to broadly treatdifferent GB patients. Applicants' present study is the first toinvestigate the utilization of CAR-modified NK-92 cells against GB.CAR-modified NK-92 cells have been shown to effectively target othercancers. For example, Esser et al. engineered NK-92 cells to stablyexpress CAR against disialoganglioside GD2, which is expressed at highlevels on neuroblastoma (NB) cells (Esser, R. et al. (2012) J Cell MolMed. 16:569-581). Expression of GD2-CAR on NK-92 cells enhanced specificelimination of GD2-expressing NB cells that were resistant to killing byparental NK-92 cells, demonstrating the potential clinical utility ofthe GD2-CAR NK cells against NB. Applicants have recently developedCS1-CAR-expressing NK-92 cells to target multiple myeloma (MM) cells anddemonstrated that these engineered NK-92 cells were able to displayenhanced cytolysis and IFN-γ production when co-cultured with MM cellsin vitro (Chu, J. et al. (2014) Leukemia 28:917-927). Importantly, in anaggressive orthotopic MM xenograft mouse model, adoptive transfer ofCS1-CAR-modified NK-92 cells efficiently impeded the dissemination ofhuman IM9 MM cells in vivo and also significantly prolonged the overallsurvival of IM9-bearing mice (Chu, J. et al. (2014) Leukemia28:917-927).

To Applicants' knowledge, this is the first study investigating thepotential of CAR immune cells to target both wtEGFR and EGFRvIII for GBtreatment. Applicants generated EGFR-CAR-engineered NK-92 and NKL cellsas well as primary NK cells which in vitro efficiently lyse GB cellsexpressing either wtEGFR or EGFRvIII. Both molecules play contributoryroles in gliomagenesis. EGFRvIII-expressing cells typically co-existwith wtEGFR-expressing cells in same patients. Biernat reported that 40%(8 out of 20) of EGFR⁺ GB biopsies also showed positive for EGFRvIII,but only 15% (3 out of 20) with a predominant EGFRvIII amplification,suggesting that the limited effectiveness could only be achieved fortherapeutic approaches based on selective targeting of EGFRvIII(Biernat, W. et al. (2004) Brain Pathol. 14:131-136). In another study,among 58 GB tumors, 83% (48/58) stained for wtEGFR by THC Furthermore,19% (11/58) of these samples were double positive for wtEGFR andEGFRvIII (Fan, Q. W. et al. (2013) Cancer Cell 24:438-449). These twostudies demonstrate that a substantial portion of GB patients havewtEGFR-expressing and EGFRvIII-expressing cells. Thus, compared withtherapeutic approaches to treat GB selectively targeting either wtEGFRor EGFRvIII, the EGFR-CAR NK therapy described here targets both wtEGFRand EGFRvIII. As noted above, EGFRvIII-specific CAR T cells show goodanti-tumor activity against GB expressing EGFRvIII both in vitro and invivo (Morgan, R. A. et al. (2012) Hum Gene Ther. 23:1043-1053; Ohno, M.et al. (2010) Cancer Sci. 101:2518-2524: Sampson, J. H. et al. (2014)Clin Cancer Res. 20:972-984). Applicants believe that an approachtargeting both wtEGFR and EGFRvIII should be effective to treat not onlyGB patients with wtEGFR-expressing tumor cells alone, but also GBpatients with both wtEGFR-expressing tumor cells and EGFRvIII-expressingtumor cells. For this purpose, Applicants engineered NK-92 cells toexpress an EGFR-CAR targeting both wtEGFR and EGFRvIII, and demonstratedthat GB cells expressing wtEGFR or EGFRvIII could be efficientlyeliminated in vitro and/or in vivo.

A potential challenge of targeting wtEGFR as well as EGFRvIII on gliomacells is the presence of wtEGFR on normal tissues. Systemic infusion ofEGFRvIII-specific CAR T cells is believed to be relatively safe, asEGFRvIII is not expressed on normal epithelial cells that can expresshigh levels of wtEGFR. However, systemic administration ofNK-92-EGFR-CAR or primary NK cells could result in serious systemictoxicity due to their impact on normal wtEGFR-expressing cells. Tominimize this risk, Applicants injected NK-92-EGFR-CAR cellsintracranially to restrict their mobility, and demonstrated that theseCAR cells remained undetectable in tissues other than brain. In furthersupport of this strategy, a recent study shows that intracranialdelivery of toxin-conjugated scFv from an EGFR antibody targeting bothwtEGFR and EGFRvIII results in strong anti-neoplastic effects againstintracranial glioblastoma xenografts expressing wtEGFR or co-expressingwtEGFR and EGFRvIII. This local administration did not cause obvioussystemic toxicity (Chandramohan, V. et al. (2013) Clin Cancer Res.19:4717-4727). In addition, Applicants' H&E staining also showed thatNK-92-EGFR-CAR cells injected into implanted tumors in the brain residein the tumor area. However, as NK-92 is a lymphoma cell line, for futureclinical applications, NK-92 cells armed with CAR should be irradiatedprior to infusion into patients. Several preclinical studiesdemonstrated that NK-92-CAR cells received 10 Gy irradiation showedsimilar effects in vitro and in vivo when compared with non-irradiatedcells (Uherek, C. et al. (2002) Blood 100: 1265-1273; Schonfeld, K etal. (2015) Mol Ther. 23:330-338).

Alternatively, EGFR-CAR primary NK cells should be considered, asApplicants showed they are also potent to eradicate GB cells.Importantly, genetically-engineered artificial antigen-presenting cells(aAPCs) expressing membrane-bound IL-15 (mbIL15) or membrane-bound IL-21(mbIL21) have recently been used to successfully expand primary NK cells(Jordan, C. T. (2009) Cell Stem Cell 4:203-205; Brennan, C. et al.(2009) PLoS One 4:e7752).

Importantly, in Applicants' study Applicants observed high expression ofendogenous EGFRvIII on the majority of patient-derived GB stem cellstested, making them more susceptible to lysis by NK-92-EGFR-CAR cellsthan by NK-92 cells transduced with control vector. GB stem cells are asubpopulation of tumor cells that reflect biological and pathologicalcharacteristics of primary GB, and retain the capability to undergoself-renewal, multi-lineage differentiation, and regeneration of theentire tumor population (Jordan, C. T. (2009) Cell Stem Cell 4:203-205).GB stem cells are considered to be responsible for tumor initiation,propagation, recurrence, and chemo- and radio-resistance. Targeting GBstem cells is believed to be a key aspect in the prevention of GBrelapse. In fact, Applicants' in vivo data show that one timeadministration of NK-92-EGFR-CAR cells significantly prolongs thesurvival of mice implanted with patient-derived GB stem cells expressingEGFRvIII. Thus, Applicants believe Applicants' study demonstrating theuse of NK-92-EGFR-CAR cells to target GB cells and GB stem cells both invitro and in two xenograft mouse models is highly relevant to thepotential treatment of relapsed human GB in the clinic.

GB is composed of distinct molecular subtypes. Mesenchymal (MES) andproneural (PN) GBs have been identified as the most important subtypesof GB (Mao, P. et al. (2013) Proc Natl Acad Sci. USA 110:8644-8649;Brennan, C. et al. (2009) PLoS One 4:e7752; Phillips, H. S. et al.(2006) Cancer Cell 9:157-173; Verhaak, R. G. et al. (2010) Cancer Cell17:98-110). In Applicants' experiments, all MES and two of three PN GBstem cells (GSCs) that Applicants tested were positive for EGFRexpression by flow cytometry. PCR analysis further demonstrated that allof these GB stem cells express EGFRvIII, while GB non-stem cell linesusually express only wtEGFR (except for ectopic over-expression ofEGFRvIII in Gli36dEGFR). Consistent with these findings, Applicants' invitro data demonstrate that EGFR-CAR NK cells display enhanced IFN-γsecretion and cytotoxicity when cultured with either PN or MES EGFR⁺GSCs in vitro. MES GSCs are more aggressive in vitro and are capable ofquickly giving rise to intracranial xenografts in vivo (Mao, P. et al.(2013) Proc Natl Acad Sci. USA 110:8644-8649) MES GSCs also exhibitremarkable resistance to radiation compared with PN GSCs (Mao, P. et al.(2013) Proc Natl Acad Sci. USA 110:8644-8649). Applicants' in vivo datademonstrated that the NK-92-EGFR-CAR cells showed efficacy in theaggressive GB30 MES GSC xenograft model.

In conclusion, Applicants have successfully generated CAR NK cells thattarget both wtEGFR and EGFRvIII in GB. NK cells armed with this EGFR-CARefficiently and specifically recognize and eradicate GB cells and/ortheir stem cells in vitro and/or in vivo. Applicants' study supports theclinical application of EGFR-CAR-modified NK-92 cells for the treatmentof relapsed GB, which may be locally administered alone or incombination with other approaches.

Experiment 2—Combination of EGFR CAR Expressing Cells and oHSV CellCulture

Human breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7, aswell as 293T and Phoenix cells, were cultured in DMEM (Invitrogen, GrandIsland, N.Y.) and supplemented with 10% FBS, penicillin (100 U/ml), andstreptomycin (100 μg/ml) (all from Invitrogen). Human NK cell line NK-92and primary NK cells (obtained from the American Red Cross in Columbus)were maintained in RPMI-1640 (Invitrogen) supplemented with 20% FBS,penicillin (100 U/ml), streptomycin (100 μg/ml), and 200 IU/mLrecombinant human (rh) IL-2 (Gold Biotechnology, MO).

Mice

Six to eight-week-old NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ (NSG) micewere obtained from Jackson Laboratories (Bar Harbor, Me.). All animalwork was approved by The Ohio State University Animal Care and UseCommittee. Mice were monitored daily for disease progression andsacrificed when they became moribund with neurologic impairments orshowed obvious weight loss.

Generation of EGFR-CAR Lentiviral Construct

The anti-EGFR single chain variable fragment (scFv) was derived from DNAsequences encoding a specific monoclonal antibody against both wtEGFRand EGFRvIII. See Soffietti et al. (2002) J. Neurol. 249(10):1357-1369.The VH-linker-VL fragment was incorporated in frame with the CD28-CD3ζportion incised from a retroviral vector. The entireanti-EGFR-scFv-CD28-CD3ζ fragment was then ligated into a lentiviralvector designated as pCDH-CMV-MCS-EF1-copGFP (System Biosciences,Mountain View, Calif.) to generate the pCDH-EGFR-scFv-CD28-CD3ζ(pCDH-EGFR-CAR) construct.

Lentiviral Production and Transduction of NK-92 Cells:

To produce lentivirus for infection of NK-92 cells, 293T cells wereco-transfected with the aforementioned pCDH-EGFR-scFv-CD28-CD3ζ plasmidor a mock pCDH vector together with packaging constructs pCMV-VSVG andpCMV-DR9 using calcium phosphate transfection reagent (Promega, Madison,Wis.). The transfection and infection procedures were modified from theprotocol of Chu et al. (2014) Official Journal of the AACR,20(15):3989-4000.

Generation of MDA-MB-231 Cells Stably Expressing CBRluc-EGFP:

MDA-MB-231 cells stably expressing CBRluc-EGFP was generated byretroviral transfection with the ΔU3CBRluc-EGFP vector (a generous giftfrom Dr. J F DiPersio) following the Chu et al. (2014) protocol. EGFPpositive breast cancer cells were then sorted using a FACS Aria II cellsorter (BD Biosciences, San Jose, Calif.) and expanded, yieldingMDA-MB-231-CBRluc-EGFP cells.

Flow Cytometry Analysis:

To determine EGFR expression on the surface of breast cancer cell lines,cells were incubated with the mouse monoclonal anti-human EGFR (cloneH11, DAKO) antibody, followed by staining with APC-conjugated goatanti-mouse IgG secondary antibody. The surface expression of EGFR-CARwas assessed by flow cytometry as described in Chu et al. (2014).

Hematoxylin and Eosin (HE) Staining and Immunohistochemistry (IHC)Assay:

Paraffin-embedded sections of tumor tissues from patients with bothprimary breast cancer and brain metastasis were stained with HE or withanti-wild-type-EGFR antibody (1:2000, DAK-H1-WT; Agilent Technologies,Santa Clara, Calif.) for IHC. An automatic immunostainer (BenchMark XT,Ventana Medical Systems, Tucson, Ariz.) was used according to themanufacturer's instructions. Sections were visualized and photographedby a Leica laser confocal microscope (SP5Wetzlar, Germany).

Cytotoxicity Assay

A standard 4-h ⁵¹Cr release assay was performed as described previously(Yu, J. et al. (2010) Blood 115:274-281). The percentage of specificcell lysis was calculated using the standard formula: 100×(cpmexperimental release−cpm spontaneous release)/(cpm maximal release−cpmspontaneous release).

IFN-γ Release Assay

1×10⁶ target cells were incubated with equal numbers of effector cellsin the wells of 96-well V-bottom plates for 24 h. Cell-free supernatantswere assayed for IFN-γ secretion by enzyme-linked immunosorbent assay(ELISA) using a kit from R&D Systems (Minneapolis, Minn.) according tothe manufacturer's protocol. Data depicted in figures represent meanvalues of triplicate wells from one of three representative experimentswith similar results.

MTS Assay

Breast cancer cell line cells (5×10³) were seeded in 96-well flat bottomculture plates and incubated at 37° C. in DMEM medium containing 10%FBS. At the end of treatment, cell viability was determined using arapid, tetrazolium-based MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt) colorimetric assay (CellTiter 96 cell proliferation assaykit; Promega, Madison, Wis.) according to the manufacturer'sinstructions. See Hayon et al. (2003) Leuk. Lymphoma 44(11):1957-1962.All experiments were performed at least in triplicates on three separateoccasions.

Luciferase Assay

MDA-MB-231-CBRluc-EGFP cells (5×10³) were seeded in 96-well flat bottomculture plates and incubated at 37° C. in DMEM medium containing 10% FBSwith different treatments. At different time points, 20 μL of theculture media were collected directly for luciferase assays using theDual-Glo Luciferase Assay System (Promega), as described in Fu et al.(2010) PLoS One. 5(7):e11867. At day 4, cell pellets were rinsed twicewith PBS, and then lysed with 30 μL of 1× passive lysis buffer(Promega). Lysates were pelleted by centrifugation (13,000 rpm, 1minute) and the supernatant was collected to measure luciferaseactivity.

Treatment of Breast Cancer Brain Invasion in NSG Mice

NSG mice were anesthetized and fixed in a stereotactic apparatus, and1×10⁵ MDA-MB-231-CBRluc-EGFP cells in 2 μL Hank's buffered salt solution(HBSS) were injected into mouse brain on day 0, where a burr hole wasdrilled 2 mm laterally and 1 mm anteriorly to the right bregma to adepth of 3.25 mm. On day 10, the mice were injected intratumorally with2×10⁶ effector cells, i.e. EGFR-CAR-transduced NK-92 cells(NK-92-EGFR-CAR) or empty vector-transduced NK-92 cells (NK-92-EV) in 5μL HBSS. The oHSV-1 alone group was injected intratumorally with 2-10⁵plaque-forming units (pfu) oHSV-1 (rQNestin34.5)—see Kambara et al.(2005) Cancer Res. 65(7):2832-2839—in 5 μL HBSS. Mice treated with 5 μlHBSS were used as a control. On day 15, mice in the CAR plus oHSV-1treatment group were intratumorally injected with 2×10⁵ pfu oHSV-1. Micewere monitored daily and euthanized when they showed signs of morbidity.Four weeks after inoculation with MDA-MB-231-CBRluc-EGFP cells, the micewere intraperitoneally (i.p.) infused with D-luciferin (150 mg/kg bodyweight; Gold Biotechnology, St. Louis, Mo., USA), anesthetized withisoflurane, and imaged using the In Vivo Imaging System (IVIS-100,PerkinElmer, Waltham Mass., USA) with living image software(PerkinElmer).

Statistics

The unpaired Student's t test was used to compare two independent groupsfor continuous endpoints if normally distributed with or without datatransformation. One-way ANOVA was used to compare among three or moregroups. For survival data, Kaplan-Meier analysis was used to estimatesurvival functions and log-rank test was used to compare the survivalbetween two groups. All tests were two-sided. P values were adjusted formultiple comparisons using Holm's procedure. A P value of less than 0.05was considered statistically significant.

Results Expression of EGFR in Breast Cancer Cell Lines and Primary andMetastatic Tissues

To assess the surface expression of EGFR in breast cancer cell lines,cells were stained with an EGFR-specific antibody, followed by flowcytometric analysis. As shown in FIG. 12A, EGFR was expressed on thesurface of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines, although levelswere clearly lower on MCF-7 cells. EGFR expression was then evaluated byimmunohistochemistry (IHC) in primary tumor tissues and thecorresponding brain metastasis lesions from two cases of patientsdiagnosed with metastatic breast cancer, after confirming the existenceof tumor cells by hematoxylin and eosin (HE) staining (FIG. 12B, top tworows). Surface EGFR expression was observed not only on tumor cells fromthe primary lesions, but also on those from the brain metastases (FIG.12B, bottom two rows)

Enhanced Cytotoxicity and IFN-γ Production of EGFR-CAR NK-92 and PrimaryNK Cells

Applicants generated a second-generation EGFR-CAR construct in the pCDHlentiviral backbone. This construct sequentially contains a signalpeptide, EGFR scFv, a hinge region, CD28, and CD3ζ. NK-92 and primary NKcells from healthy donors were transduced with the CAR-expressinglentiviruses and sorted based on expression of GFP by the vector.Applicants performed flow cytometric analysis using a goat anti-mouseF(ab′)₂ antibody that recognized the scFv portion of anti-EGFR. FIG. 13Ashows the expression of EGFR-CAR on the surface of EGFR-CAR-transducedNK-92 cells, which was undetectable on NK-92-EV cells (NK-92 cellstransduced with the empty vector pCDH). Next, Applicants exploredwhether EGFR-CAR expression could confer NK-92 and primary NK cells withenhanced IFN-γ production and cytolytic activity. Applicants observedthat EGFR-CAR-transduced NK-92 and primary NK cells (FIG. 18) secretedsignificantly higher levels of IFN-γ when co-cultured with MDA-MB-231cells or MDA-MB-468 cells as compared to their corresponding effectorcells transduced with an empty vector (FIGS. 13B, 14A). Interestingly,this change in IFN-γ secretion was less discernible when MCF-7 cellswith a lower level of EGFR expression were used as targets. Moreover,upon co-culture with these three cell lines, Applicants observed asignificant increase in the cytotoxic activity of EGFR-CAR-transducedNK-92 and primary donor derived NK cells compared to that ofmock-transduced NK-92 effector cells (FIGS. 13C-13E) or primary NKcells, respectively (FIGS. 14B-14D). Using CD69 surface expression tomeasure effector cell activation, Applicants also observed that tumorcells with EGFR expression can activate EGFR-CAR-transduced NK-92 cells,with higher activation when MDA-MB-231 and MDA-MB-468 cells were usedthan when MCF-7 cells were used. Applicants also detected expression ofCD27, another NK cell activation marker, and observed that CD27 was notexpressed on the surface of EGFR-CAR NK-92 cells (FIG. 19)

Lysis of Breast Cancer Cell Lines by oHSV-1

Applicants explored whether oHSV-1 alone could lyse and destroy breastcancer cells, which have the capability of trafficking into the brain toform metastatic brain tumors. As shown in FIG. 15A, oHSV-1 reduced theviability of MDA-MB-231, MDA-MB-468, and MCF-7 cells in a dose-dependentfashion after co-culture for 48 h, and this effect was observed atdifferent time points (FIG. 15B). Microscopic analysis showed thatoHSV-1 alone could lyse these breast cancer cell line cells afterco-culture for 4 days (FIG. 20A). This was confirmed usingluciferase-expressing MDA-MB-231 cells (MDA-MB-231-CBRluc-EGFP), inwhich a higher level of luciferase was detected in the supernatants fromthe group with oHSV-1 infection compared to the mock-infected group(P<0.01 at day 4) (FIG. 15C). Meanwhile, oHSV-1 did not lyse or induceapoptosis of EGFR-CAR NK-92 effector cells, as determined by amicroscopic examination (FIG. 20B).

EGFR-CAR NK-92 Cells in Combination with oHSV-1 Result in More EfficientEradication of Cancer Cells In Vitro

When MDA-MB-231 cells were treated with EGFR-CAR NK-92 cells alone or incombination with oHSV-1 (either treatment with EGFR-CAR NK-92 cells for4 h followed by oHSV-1 treatment or vice versa), MTS assays indicatedthat the MDA-MB-231 cell line was efficiently killed under allcircumstances; however, the combination of EGFR-CAR NK-92 cells withoHSV-1 resulted in more efficient killing (data not shown). Applicantsthen assessed killing by measuring luciferase activity in thesupernatants of MDA-MB-231-CBRluc-EGFP cells following differenttreatments. Luciferase was found to be degraded quickly (not shown), andthus, the luciferase assay allowed us to determine dynamic, real-timekilling rather than accumulative killing. Based on this, Applicantsobserved that EGFR-CAR NK-92 cells alone and EGFR-CAR NK-92 cellscombined with oHSV-1 caused more rapid lysis than oHSV-1 alone (FIG.5A). When measuring luciferase activity in the remainingMDA-MB-231-CBRluc-EGFP cells (cell pellets) after a co-culture for 4days, Applicants found that EGFR-CAR NK-92 cells alone, oHSV-1 alone, orEGFR-CAR NK-92 cells combined with oHSV-1 all led to substantial killingof MDA-MB-231-CBRluc-EGFP cells, and EGFR-CAR NK-92 cells combined withoHSV-1 regardless of the order was more effective than the monotherapies(FIG. 16B). Similar results were observed by microscopic examination(FIG. 21). EGFR-CAR NK-92 cells quickly destroyed some of the MDA-MB-231cells, but a subset of these cells still maintained their original cellshape and integrity even after 5 days. oHSV-1 first caused the targetcancer cells to aggregate, then the cells were gradually lysed (FIG.21). However, the combination of EGFR-CAR NK-92 cells and oHSV-1resulted in more robust cell killing, especially in the CAR NK-92 cellsfollowed by oHSV-1 treatment group (row 5, FIG. 21). Of note, consistentwith ⁵¹Cr release assays (FIG. 13C) and luciferase data (FIG. 16B),microscopic analysis demonstrated that MDA-MB-231 cells were resistantto killing by NK-92-EV cells.

EGFR-CAR NK-92 Cells Combined with oHSV-1 Lead to More Efficient Killingof MDA-MB-231 Tumor Cells in an Intracranial Model

To further support the potential therapeutic application of EGFR-CARNK-92 cells, oHSV-1 alone, or the combination of both, Applicantsexamined their antitumor activity in vivo. Applicants established anintracranial model of breast cancer by implanting MDA-MB-231-CBRluc-EGFPcells into the brains of NSG mice. The expression of beetle redluciferase in the cells enabled us to monitor tumor growth via in vivobioluminescence imaging. To minimize potential systemic toxicity,Applicants injected the non-irradiated EGFR-CAR NK-92 cells or oHSV-1intratumorally at day 10 post-tumor cell implantation and oHSV-1 at day15 for the group of EGFR-CAR NK-92 combined with oHSV-1. As shown inFIG. 17A and FIG. 22, mice that received either EGFR-CAR NK-92, oHSV-1,or their combination had significantly reduced tumor growth compared tothose injected with mock-transduced NK-92-EV or vehicle (HBSS).Importantly, the reduction in tumor growth was more obvious in micetreated with EGFR-CAR NK-92 combined with oHSV-1 than in those treatedwith EGFR-CAR NK-92 alone or oHSV-1 alone. In agreement with these data,the mice treated with EGFR-CAR NK-92 plus oHSV-1 survived significantlylonger than those treated with oHSV-1 alone (P<0.01), mock-transducedNK-92 (P<0.001), or HBSS (P<0.001), while the difference between thegroup of EGFR-CAR NK-92 plus oHSV-1 and EGFR-CAR NK-92 alone showed thesame trend and was at the border of the significance threshold(P=0.0757). The median survival time of the five groups for EGFR-CARNK-92 combined with oHSV-1, EGFR-CAR NK-92, oHSV-1, NK-92-EV and HBSSwere 80, 61, 55, 43, and 42 days, respectively (FIG. 17B).

Discussion

These data demonstrate that intratumoral administration of EGFR-CARNK-92 cells, oHSV-1, or the combination of both into mice pre-inoculatedwith MDA-MB-231 cells led to antitumor efficacy and their combinationresulted in more efficient suppression of tumor growth and significantlylonger survival of tumor-bearing mice.

Applicants believe this combination is an effective approach to targetcancer. Not to be bound by theory, Applicants suspect the target of thiscombination approach are cancer stem cells (CSCs), a cell populationresponsible for relapse, treatment resistance, and metastasis in most ifnot all cancers. Further, Applicants posit that the disclosed approachmay accommodate heterogeneous tumor populations.

That is, (considering the example of breast cancer disclosed hereinabove) EGFR-expressing cells are targeted by EGFR-CAR NK cells, butoHSV-1 also can kill EGFR-negative tumor cells. Breast cancer isheterogeneous for EGFR, PR, and HER2 expression. Although the breastcancer cell lines used in our experiments express wild-type EGFR, eachexpresses this to a different degree. Meanwhile, they have differentgene expression profiling [MDA-MB-231 and MDA-MB-468: ER−, PR−, HER2−(triple negative); MCF-7: ER+, PR+/−, HER2−] and distinct biologicalbehaviors. Triple-negative breast cancer (TNBC) is associated with anaggressive natural history as well as an increased susceptibility tometastasis Patients with TNBC lack the “traditional” therapeutic targetsand have a poorer prognosis than other types of breast cancer. In fact,median survival for TNBC is only 4.9 months. The combinational approachdescribed in this above should target BCBMs of both non-TNBC and TNBC,as oHSV is effective for the general BCBM population, while EGFR-CAR NKcells are more effective in targeting the EGFR+ populations. Suchconclusions are further generalizable to essentially all type ofcancers.

These data further showed that EGFR-CAR NK-92 cells can quickly targetand attack breast cancer cells while oHSV-1 can slowly but constantlyinfect and destroy the cancer cells. EGFR-CAR NK-92 cells can usuallyrecognize and attack target cells in several hours, but they can surviveonly several days because they have to be irradiated as the cell linewas originally established from a patient with non-Hodgkin's lymphoma.An irradiation dose of 1000 cGy has been optimized to suppressproliferation of NK-92 cells while maintaining full cytotoxic activityup to 48 hours post irradiation On the contrary, it may take about 4days for oHSV to enter into target cells, replicate, and destroy thetumor cells, even though its effects can last for a long time. Inaddition, CAR-modified NK cells may destroy the tumor tissue structureand decrease the connection between tumor cells, increase thepermeability of cancer cell membranes, and therefore enhance virusdistribution and replication in cancer cells when combined with oHSV-1.

EQUIVALENTS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this technology belongs.

The present technology illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the present technologyclaimed.

Thus, it should be understood that the materials, methods, and examplesprovided here are representative of preferred aspects, are exemplary,and are not intended as limitations on the scope of the presenttechnology.

The present technology has been described broadly and genericallyherein. Each of the narrower species and sub-generic groupings fallingwithin the generic disclosure also form part of the present technology.This includes the generic description of the present technology with aproviso or negative limitation removing any subject matter from thegenus, regardless of whether or not the excised material is specificallyrecited herein.

In addition, where features or aspects of the present technology aredescribed in terms of Markush groups, those skilled in the art willrecognize that the present technology is also thereby described in termsof any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other aspects are set forth within the following claims.

1. (canceled)
 2. The method of claim 63, wherein the costimulatorymolecule comprises a molecule or polypeptide selected from the group of:a 4-1BB costimulatory signaling region, a CD28 costimulatory molecule,OX40, ICOS, CD27, CD30, CD40, PD-1, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand thatspecifically binds with CD83.
 3. The method of claim 63, wherein theantigen binding domain of the anti-EGFR antibody comprises an anti-EGFRheavy chain (HC) variable region comprising the amino acid sequence ofSEO ID NO: 24 or an equivalent thereof having at least 80% identity toSEO ID NO: 24 and an anti-EGFR light chain (LC) variable regioncomprising the amino acid sequence of SEO ID NO: 26 or an equivalentthereof having at least 80% identity to SEO ID NO:
 26. 4-5. (canceled)6. The method of claim 3, further comprising a linker polypeptidelocated between the anti-EGFR HC variable region and the anti-EGFR LCvariable region.
 7. The method of claim 6, wherein the linkerpolypeptide comprises GGGGSGGGGSGGGG, or an equivalent thereof.
 8. Themethod of claim 63, further comprising a signal polypeptide to the aminoterminus of the EGFR binding domain.
 9. The method of claim 63, whereinthe hinge polypeptide comprisesCTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG, or an equivalentthereof.
 10. The method of claim 2, wherein the CD28 costimulatorymolecule comprises a CD28 transmembrane domain and a CD28 intracellulardomain, or an equivalent of each thereof.
 11. The method of claim 63,further comprising a detectable marker and/or a purification markerattached to the CAR. 12.-40. (canceled)
 41. The method of claim 3,wherein the cell is a cell of the group consisting of a mammalian cell,a canine cell, a bovine cell, a murine cell, a feline cell, an equinecell or a human cell, and optionally a stem cell, a macrophage, a T cellor an NK cell. 42.-50. (canceled)
 51. The method of claim 63, whereinthe isolated cells are autologous to the subject being treated.
 52. Themethod of claim 63, wherein the cells are administered by intracranialinjection or intravenous administration.
 53. The method of claim 63,wherein the subject is selected from the group of a mammal, a murine, acanine, a bovine, a murine, a feline, an equine or a human. 54.-57.(canceled)
 58. The method of claim 63, further comprising administeringan effective amount of anti-cancer therapy.
 59. The method of claim 58,wherein the anti-cancer therapy comprises one or more of radiationtherapy, surgery or chemotherapy. 60.-62. (canceled)
 63. A method fortreating a cancer in a subject in need thereof, comprising administeringto the subject: an effective amount of isolated cells expressing achimeric antigen receptor (CAR) comprising (a) an antigen binding domainof an anti-Epidermal Growth Factor Receptor (anti-EGFR) antibody thatrecognizes both wild type Epidermal Growth Factor Receptor (wtEGFR) andEpidermal Growth Factor Receptor variant III mutant (EGFRvIII), whereinthe antigen binding domain comprises the three complementaritydetermining regions (CDRs) of an anti-EGFR heavy chain (HC) variableregion of SEO ID NO: 24; and the three CDRs of an anti-EGFR light chain(LC) variable region of SEO ID NO: 26: (b) a hinge domain polypeptide;(c) a costimulatory molecule or polypeptide; and (d) a CD3 zetasignaling domain; and an effective amount of an oncolytic virus.
 64. Themethod of claim 63, wherein the isolated cell is an NK cell.
 65. Themethod of claim 63, wherein the isolated cells have been irradiated. 66.The method of claim 65, wherein the isolated cells have been irradiatedat a dose of 1000 cGy.
 67. The method of claim 75, wherein the oncolyticherpes simplex virus is or is derived from an HSV-1.
 68. The method ofclaim 63, wherein the effective amount of the isolated cells isadministered before the effective amount of the oncolytic virus.
 69. Themethod of claim 68, wherein the effective amount of oncolytic virus isadministered over multiple days.
 70. The method of claim 63, wherein thecancer is a brain cancer.
 71. The method of claim 70, wherein the braincancer is selected from a glioblastoma or a breast cancer brainmetastasis.
 72. (canceled)
 73. The method of claim 63, wherein theeffective amount of the isolated cells is administered after theeffective amount of the oncolytic virus.
 74. The method of claim 63,wherein the effective amount of the isolated cells is administeredsimultaneously with the effective amount of the oncolytic virus.
 75. Themethod of claim 63, wherein the oncolytic virus is an oncolytic herpessimplex virus.
 76. A therapeutic combination comprising: an isolatedcell expressing a chimeric antigen receptor (CAR) comprising (a) anantigen binding domain of an anti-Epidermal Growth Factor Receptor(anti-EGFR) antibody that recognizes both wild type Epidermal GrowthFactor Receptor (wtEGFR) and Epidermal Growth Factor Receptor variantIII mutant (EGFRvIII), wherein the antigen binding domain comprises thethree complementarity determining regions (CDRs) of an anti-EGFR heavychain (HC) variable region of SEQ ID NO: 24 and the three CDRs of ananti-EGFR light chain (LC) variable region of SEQ ID NO: 26; (b) a hingedomain polypeptide; (c) a costimulatory molecule or polypeptide; and (d)a CD3 zeta signaling domain; and an oncolytic virus.
 77. The therapeuticcombination of claim 76, wherein the costimulatory molecule comprises amolecule or polypeptide selected from the group of: a 4-1BBcostimulatory signaling region, a CD28 costimulatory molecule, OX40,ICOS and CD27.
 78. The therapeutic combination of claim 76, wherein theantigen binding domain of the anti-EGFR antibody comprises an anti-EGFRheavy chain (HC) variable region comprising the amino acid sequence ofSEQ ID NO: 24 or an equivalent thereof having at least 80% identity toSEQ ID NO: 24; and an anti-EGFR light chain (LC) variable regioncomprising the amino acid sequence of SEQ ID NO: 26 or an equivalentthereof having at least 80% identity to SEQ ID NO:
 26. 79. Thetherapeutic combination of claim 77, wherein the CD28 costimulatorymolecule comprises a CD28 transmembrane domain and a CD28 intracellulardomain.
 80. The therapeutic combination of claim 76, wherein theoncolytic virus is an oncolytic herpes simplex virus.
 81. Thetherapeutic combination of claim 80, wherein the oncolytic herpessimplex virus is or is derived from an HSV-1.
 82. The therapeuticcombination of claim 76, wherein the isolated cell is an NK cell. 83.The therapeutic combination of claim 76, wherein the therapeuticcombination is formulated to treat a brain cancer.
 84. The therapeuticcombination of claim 83, wherein the brain cancer is selected from aglioblastoma or a breast cancer brain metastasis.
 85. The therapeuticcombination of claim 76, wherein the isolated cell is formulated forintracranial injection or intravenous administration.